Preparation of lactams from aliphatic α, ω-dinitriles

ABSTRACT

A process for the preparation of five-membered or six-membered ring lactams from aliphatic α,ω-dinitriles has been developed. In the process an aliphatic α,ω-dinitrile is first converted to an ammonium salt of an ω-nitrile-carboxylic acid in aqueous solution using a catalyst having an aliphatic nitrilase (EC 3.5.5.7) activity, or a combination of nitrile hydratase (EC 4.2.1.84) and arnidase (EC 3.5.1.4) activities. The ammonium salt of the ω-nitrilecarboxylic acid is then converted directly to the corresponding lactam by hydrogenation in aqueous solution, without isolation of the intermediate ω-nitrilecarboxylic acid or ω-aminocarboxylic acid. When the aliphatic α,ω-dinitrile is also unsymmetrically substituted at the α-carbon atom, the nitrilase produces the ω-nitrilecarboxylic acid ammonium salt resulting from hydrolysis of the ω-nitrile group with greater than 98% regioselectivity, thereby producing only one of the two possible lactam products during the subsequent hydrogenation. A heat-treatment process to select for desirable regioselective nitrilase or nitrile hydratase activities while destroying undesirable activities is also provided.

This is a division of application Ser. No. 08/650,073 filed May 17,1996, which issued as U.S. Pat. No. 5,858,736 on Jan. 12, 1996.

FIELD OF THE INVENTION

This invention relates to a process for the preparation of five-memberedor six-membered ring lactams from aliphatic α,ω-dinitriles by acombination of biological and chemical techniques. More particularly, analiphatic α,ω-dinitrile is first converted to an ammonium salt of anω-nitrilecarboxylic acid in aqueous solution using a catalyst having analiphatic nitrilase (EC 3.5.5.7) activity, or a combination of nitrilehydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) activities. Theammonium salt of the ω-nitrilecarboxylic acid is then converted directlyto the corresponding lactam by hydrogenation in aqueous solution,without isolation of the intermediate ω-nitrilecarboxylic acid orω-aminocarboxylic acid. When the aliphatic α,ω-dinitrile is alsounsymmetrically substituted at the α-carbon atom, the nitrilase producesthe ω-nitrilecarboxylic acid ammonium salt resulting from hydrolysis ofthe ω-nitrile group with greater than 98% regioselectivity, therebyproducing only one of the two possible lactam products during thesubsequent hydrogenation.

DESCRIPTION OF THE RELATED ART

Nitriles are readily converted to the corresponding carboxylic acids bya variety of chemical processes, but these processes typically requirestrongly acidic or basic reaction conditions and high reactiontemperatures, and usually produce unwanted byproducts and/or largeamounts of inorganic salts as unwanted byproducts. Processes in whichenzyme-catalyzed hydrolysis convert nitrile substrates to thecorresponding carboxylic acids are often preferred to chemical methods,since these processes are often run at ambient temperature, do notrequire the use of strongly acidic or basic reaction conditions, and donot produce large amounts of unwanted byproducts. An additionaladvantage of the enzyme-catalyzed hydrolysis of nitriles over chemicalhydrolysis is that, for the hydrolysis of a variety of aliphatic oraromatic dinitriles, the enzyme-catalyzed reaction can be highlyregioselective, where only one of the two nitrile groups is hydrolyzedto the corresponding carboxylic acid ammonium salt.

A nitrilase enzyme directly converts a nitrile to the correspondingcarboxylic acid ammonium salt in aqueous solution without theintermediate formation of an amide. The use of aromatic nitrilases forthe hydrolysis of aromatic nitrites to the corresponding carboxylic acidammonium salts has been known for many years, but it is only recentlythat the use of aliphatic nitrilases have been reported. Kobayashi etal. (Tetrahedron, (1990) vol. 46, 5587-5590; J. Bacteriology, (1990),vol. 172, 4807-4815) have described an aliphatic nitrilase isolated fromRhodococcus rhodochrousK 22 which catalyzed the hydrolysis of aliphaticnitrites to the corresponding carboxylic acid ammonium salts, severalaliphatic (α,ω-dinitriles were also hydrolyzed, and glutaronitrile wasconverted to 4-cyanobutyric acid ammonium salt with 100% molarconversion using resting cells as catalyst. A nitrilase from Comcimonastestosteroni has been isolated which can convert a range of aliphaticα,ω-dinitriles to either the corresponding ω-nitrilecarboxylic acidammonium salt or the dicarboxylic acid diammonium salt (Canadian patentapplication CA 2,103,616 (1994/02/11); S. Levy-Schil, et al., Gene,(1995), vol. 161, 15-20); for the hydrolysis of adiponitrile, a maximumyield of 5-cyanovaleric acid ammonium salt of ca. 88% was obtained priorto complete conversion of the 5-cyanovaleric acid ammonium salt toadipic acid diammonium salt.

M. L. Gradley and C. J. Knowles (Biotechnology Lett., (1994), vol. 16,41-46) have reported the use of suspensions of Rhodococcus rhodochrousNCIMB 11216 having an aliphatic nitrilase activity for the hydrolysis ofseveral 2-methylalkylnitriles. Complete conversion of(+/-)-2-methylbutyronitrile to 2-methylbutyric acid ammonium salt wasobtained, while the hydrolysis of (+/-)-2-methylhexanenitrile appearedto be enantiospecific for the (+)-enantiomer. C. Bengis-Garber and A. L.Gutman (AppL. MicrobioL Biotechnol., (1989), vol. 32, 11-16) have usedRhodococcus rhodochrous NCIMB 11216 as catalyst for the hydrolysis ofseveral dinitriles. In this work, fumaronitrile and succinonitrile wereconverted to the corresponding ω-nitrilecarboxylic acid ammonium salts,while glutaronitrile, adiponitrile, and pimelonitrile were converted tothe corresponding dicarboxylic acid diammonium salts.

A combination of two enzymes, nitrile hydratase (NHase) and amidase, canbe also be used to convert aliphatic nitrites to the correspondingcarboxylic acid ammonium salts in aqueous solution. Here the aliphaticnitrile is initially converted to an amide by the nitrile hydratase andthen the amide is subsequently converted by the amidase to thecorresponding carboxylic acid ammonium salt. A wide variety of bacterialgenera are known to possess a diverse spectrum of nitrile hydratase andamidase activities, including Rhodococcus, Pseudomonas, Alcaligenes,Arthrobacter, Bacillus, Bacteridium, Brevibacterium, Corynebacterium,and Micrococcus. Both aqueous suspensions of these microorganisms andthe isolated enzymes have been used to convert nitrites to amides andcarboxylic acid ammonium salts.

P. Honicke-Schmidt and M. P. Schneider (J. Chem. Soc., Chem. Commun.,(1990), 648-650) have used immobilized Rhodococcus sp. strain CH 5 toconvert nitrites and dinitriles to carboxylic acid ammonium salts andω-nitrilecarboxylic acid ammonium salts, respectively. The cells containboth a nitrile hydratase and amidase activity which convertsglutaronitrile to 4-cyanobutyric acid ammonium salt in 79% isolatedyield based on 92% conversion ot substrate. A. J. Blakely et al. (FEMSMicrobiology Lett., (1995), vol, 129. 57-62) have used the nitrilehydratase and amidase activity of suspensions of Rhoclococcus AJ270 toregiospecifically hydrolyze malononitrile and adiponitrile to produceonly the corresponding ω-nitrilecarboxylic acid ammonium salts. H.Yamada et al. (J. Ferment. Technol., (1980), vol. 58, 495-500) describethe hydrolysis of glutaronitrile to a mixture of 4-cyanobutyramide,4-cyanobutyric acid, glutaric acid and ammonia using Pseudomonas sp. K9,which contains both a nitrile hydratase and amidase. K. Yamamoto et al.(J. Ferment. Bioengineering, 1992, vol. 73, 125-129) described the useof Corynebacterium sp. CH 5 cells containing both a nitrile hydrataseand amidase activity to convert trans-1,4-dicyanocyclohexane totrans-4-cyanocyclohexanecarboxylic acid ammonium salt in 99.4% yield.

J. L. Moreau et al. (Biocatalysis, (1994), vol 10. 325-340) describe thehydrolysis of adiponitrile to adipic acid, adipamide, and adipamic acidthrough the intermediate formation of 5-cyanovaleric acid usingBrevibacterium sp. R312 (nitrile hydratase and amidase activity). A.Kerridge et al. (Biorg. Medicinal Chem., (1994), vol. 2, 447-455) reportthe use of Brevibacterium sp. R312 (nitrile hydratase and amidaseactivity) to hydrolyze prochiral 3-hydroxyglutaronitrile derivatives tothe corresponding (S)-cyanoacid ammonium salts. European Patent 178,106B1 (Mar 31, 1993) discloses selective transformation of one of the cyanogroups of an aliphatic dinitrile to the corresponding carboxylic acid,amide, ester or thioester using the mononitrilase activity (defined aseither nitrilase or a combination of nitrile hydratase/amidase) derivedfrom Bacillus, Bacteridium, Micrococcus or Brevibacterium. In additionto the many examples of bacterial catalysts having nitrilase activity ornitrile hydratase/amidase activity, Y. Asano et al. (Agric. Biol. Chem.,(1980), vol. 44, 2497-2498) demonstrated that the fugus Fusariummerismoides TG-1 hydrolyzed glutaronitrile to 4-cyanobutyric acidammonium salt, and 2-methylglutaronitrile to 4-cyanopentanoic acidammonium salt.

No prior art has been found which describes the hydrogenation ofammonium salts of aliphatic ω-nitrilecarboxylic acids in aqueoussolution to directly produce the corresponding lactams. In closelyrelated art, U.S. Pat. No. 4,329,498 describes the hydrogenation ofmuconic acid mononitrile to 6-aminocaproic acid (6-ACA) in dry ethanolsaturated with ammonia, using a Raney nickel catalyst #2. After removalof the hydrogenation catalyst, heating the ethanolic solution of 6-ACAto 170° C.-200° C. was expected to result in the cyclization of 6-ACA tocaprolactam. The reductive cyclization of either β-quinoxalinylpropanoicacids (E. C. Taylor et al., J. Am. Chem. Soc., (1965), vol. 87.1984-1990). or the related 2-(2-carboxyethyl)-3(4H)-quinoxalone (E. C.Taylor et al., J. Am. Chem. Soc., (1965). vol. 87. 1990-1995) byhydrogenation in 1 Nsodium hydroxide solution using Raney nickel as thecatalyst has been reported to produce the corresponding five-memberedring lactams, but only after removal of the catalyst from the productmixture and acidification of the resulting filtrate. The authors statethat for any of these reductions, "lactam formation can only proceed inacidic solution" (page 1992, second paragraph), presumably requiring thepresence of the protonated carboxylic acid and not the carboxylate salt.U.S. Pat. No. 4,730,040 discloses a process for the preparation ofcaprolactam, reacting an aqueous solution of 5-formylvaleric acid withammonia and hydrogen in the presence of a hydrogenation catalyst,following which ammonia is separated from the product mixture and theresulting solution of 6-ACA is heated to 300° C.

Previous work has disclosed single cells containing both nitrilehydratase and arnidase activities that have been used to convertnitriles and dinitriles to various acid ammonium salts. However, noprior art has been found which describes the cyclization of ammoniumsalts of aliphatic ω-aminocarboxylic acids under the hydrogenationreaction conditions of the present invention (i.e., in an aqueoussolution containing an excess of added ammonium hydroxide) to producethe corresponding lactams. In closely related art, the cyclization ofaliphatic ω-aminocarboxylic acids (but not the ammonium salts) to thecorresponding lactams under a variety of reaction conditions has beenreported. F. Mares and D. Sheehan (Ind. Eng. Chem. Process Des. Dev.,(1978), vol. 17, 9-16) have described the cyclization of 6-aminocaproicacid (6-ACA) to caprolactam using water or ethanol as solvent. In water,the cyclization reaction was reversible at concentrations below 1 mol/kg(ca. 1M), and the concentration of caprolactam increased with increasingtemperature; at a total concentration of 6-ACA and caprolactam of 0.85mollkg (ca. 0.85M), the percentage of caprolactam was reported toincrease from 38.7% at 180° C. to 92.2% at 250° C. In ethanol, a 98%yield of caprolactam was obtained at 200° C., reportedly due to a shiftin the equilibrium which favors the free-acid/free-amine form of 6-ACAin ethanol, rather than the intramolecular alkylammonium carboxylateform of 6-ACA which predominates in water. A process for the productionof caprolactam from 6-ACA is also described in U.S. Pat. No. 4,599,199,where 6-ACA is introduced into a fluidized alumina bed in the presenceof steam at from 290° C. to 400° C. The synthesis of five-, six- andseven-membered ring lactams by cyclodehydration of aliphaticω-aminoacids on alumina or silica gel in toluene, and with continuousremoval of the water produced during the reaction, has been reported byA. Blade-Font (Tetrahedron Letters, (1980), vol. 21, 2443-2446). A freeamino group (unprotonated) was reported to be necessary forcyclodehydration to take place.

No prior art has been found which describes the hydrogenation ofammonium salts of aliphatic ω-nitrilecarboxylic acids in aqueoussolution containing methylamine to directly produce the correspondingN-methyl lactams. In closely related art, 1,5-dimethyl-2-pyrrolidinonewas prepared by the hydrogenation of an aqueous solution of levulinicacid and methylamine in water using a Raney nickel catalyst at 140° C.and 1000-2000 psig of hydrogen R. L. Frank et al., Org. Sytheses,(1954), Coll. Vol. 3, 328-329). The resulting 4-N-methylaminopentanoicacid methylammonium salt was then cyclized to the corresponding lactarnby filtration of the product mixture and distillation of the filtrate toremove water and methylamine. N-alkyl lactams have also been produced bythe direct hydrogenation of an aqueous mixture containing2-methylglutaronitrile, a primary alkylamine, and a hydrogenationcatalyst, the process yielding a mixture of 1,3- and1,5-dialkylpiperidone-2 (U.S. Pat. No. 5,449,780). N-Substituted2-pyrrolidinones have been prepared by the reaction of γ-valerolactonewith an alkyl amine at 110-130° C., then heating the resulting mixtureto 250-270° C. while distilling off water (F. B. Zienty and G. W.Steahly, J. Am. Chem. Soc., (1947), vol. 69, 715-716).

The above processes for the production of lactams or N-alkyllactamssuffer from one or more of the following disadvantages: the use oftemperatures in excess of 250° C. to obtain high yields of lactams whenusing water as a solvent, the removal of water from the reaction mixtureto drive the equilibrium toward lactarn formation, the adjustment of thepH of the reaction mixture to an acidic value to favor lactam formation,or the use of an organic solvent in which the starting material issparingly soluble. Many of these processes generate undesirable wastestreams, or mixtures of products which are not easily separated. Asignificant advance would be a process for the conversion of analiphatic α,ω-dinitrile to the corresponding lactam or N-methyllactam inaqueous solution, in high yield with high regioselectivity, with littlebyproduct or waste stream production, and with a facile method ofproduct recovery.

SUMMARY OF THE INVENTION

A process for the preparation of five-membered ring lactams orsix-membered ring lactams from aliphatic α,ω-dinitriles, having thesteps:

(a) contacting an aliphatic α,ω-dinitrile in an aqueous reaction mixturewith an enzyme catalyst characterized by either

(1) an aliphatic nitrilase activity, or

(2) a combination of nitrile hydratase and amidase activities, wherebythe aliphatic α,ω-dinitrile is converted to an ω-nitrilecarboxylic acidammonium salt.

(b) contacting the aqueous product mixture resulting from step (a) withhydrogen and a hydrogenation catalyst, whereby the ω-nitrile carboxylicacid ammonium salt is converted directly to the corresponding lactamwithout isolation of the intermediate ω-nitrilecarboxylic acid,ω-nitrilecarboxylic acid, ammonium salt, ω-aminocarboxylic acid, orω-aminocarboxylic acid ammonium salt; and

(c) recovering the lactam from the aqueous product mixture resultingfrom step (b).

Prior to step (b), ammonium hydroxide, ammonia gas, or methylamine maybe added to the aqueous product mixture of step (a). This addition maybe from 0 to 4 molar equivalents relative to the amount ofω-nitrilecarboxylic acid ammonium salt present.

A further embodiment of the invention is a method for treating a wholecell catalyst to select for a regioselective nitrilase activity ornitrile hydratase activity capable of catalyzing the conversion ofaliphatic α,ω-dinitriles to the corresponding ω-cyanocarboxylic acidammonium salt. The whole cell catalyst to be treated is characterized bytwo types of activities: (1) a desirable regioselective nitrilaseactivity or regioselective nitrile hydratase activity and (2) anundesirable non-regioselective nitrilase or nitrite hydratase activity.Treatment of the cell involves heating the whole cell catalyst to atemperature of about 35° C. to 70° C. for between 10 and 120 minuteswherein the undesirable non-regioselective nitrilase activity or nitrilehydratase activity is destroyed and the desirable regioselectivenitrilase or nitrile hydratase activity is preserved.

Further embodiments of the invention use enzyme catalysts in the form ofwhole microbial cells, permeabilized microbial cells, one or more cellcomponents of a microbial cell extract, and partially purifiedenzyme(s), or purified enzyme(s). These enzyme catalysts can beimmobilized on a support. Microorganisms which are characterized by analiphatic nitrilase activity and useful in the process are Acidovoraxfacilis 72-PF-15 (ATCC 55747), Acidovorax facilis 72-PF-17 (ATCC 55745),and Acidovorax facilis 72 W (ACC 55746). A microorganism characterizedby a combination of nitrile hydratase and amidase activities and usefulin the process is Comomonas testosteroni 5-MGAM-4 D (ATCC 55744).

BRIEF DESCRIPTION OF THE BIOLOGICAL DEPOSITS

Applicants have made the following biological deposits under the termsof the Budapest Treaty:

    ______________________________________    Depositor Identification                       Int'l. Depository                                   Date of    Reference          Designation Deposit    ______________________________________    Comomonas testosteroni 5-MGAM-4D                       ATCC 55744  Mar. 8, 1996    Acidovorax facilis 72-PF-17                       ATCC 55745  Mar. 8, 1996    Acidovorax facilis 72W                       ATCC 55746  Mar. 8, 1996    Acidovorox facilis 72-PF-15                       ATCC 55747  Mar. 8, 1996    ______________________________________

As used herein, "ATCC" refers to the American Type Culture Collectioninternational depository located as 12301 Parklawn Drive, Rockville, Md.20852 U.S.A. The "ATCC No." is the accession number to cultures ondeposit with the ATCC.

DETAILED DESCRIPTION OF THE INVENTION

A process to prepare lactams from aliphatic α,ω-dinitriles in highyields has been developed which utilizes a combination of enzymatic andchemical reactions. In cases where the α,ω-dinitrile is unsymmetricallysubstituted, high regioselectivity to one of two possible lactamproducts (Scheme 1) is seen. ##STR1## n=1,2 R=H, unsubstituted orsubstituted alkyl, or unsubstituted or substituted alkenyl;

R¹ =H, --CH₃

The products of the present invention are useful as precursors forpolymers, solvents, and chemicals of high value in the agricultural andpharmaceutical industries. The process uses temperatures less than 250°C. to obtain a high yield of lactam when using water as a solvent.Relative to previously known chemical lactam processes, the claimedinvention generates little waste and permits a facile approach toproduct recovery.

In the application, unless specifically stated otherwise, the followingabbreviations and definitions apply:

"Enzyme catalyst" refers to a catalyst which is characterized by eithera nitrilase activity or a combination of a nitrile hydratase activityand an amidase activity. The catalyst may be in the form of a wholemicrobial cell, permeabilized microbial cell(s), one or more cellcomponent of a microbial cell extract, partially purified enzyme(s), orpurified enzyme(s).

"Hydrogenation catalyst" refers to a material that accelerateshydrogenation without itself being consumed or undergoing a chemicalchange.

"Aqueous product mixture" is used to refer to an aqueous mixturecontaining a product resulting from the corresponding process step.

A. Conversion of an aliphatic α,ω-dinitrile to the correspondingω-nitrilecarboxylic acid ammonium salt in high yield and with highregioselectivity.

The first step of this process is the conversion of an aliphaticα,ω-dinitrile to the corresponding ω-nitrilecarboxylic acid ammoniumsalt, using an enzyme catalyst. The enzyme catalyst has either anitrilase activity, where the nitrilase converts the α,ω-dinitriledirectly to a corresponding ω-nitrilecarboxylic acid ammonium salt (eqn.1), or a combination of two enzyme activities, nitrile hydratase (NHase)and amidase, where the aliphatic α,ω-dinitrile is initially converted toa ω-nitrilealkylamide by the nitrile hydratase, and then theω-nitrilealkylamide is subsequently converted by the amidase to thecorresponding ω-nitrilecarboxylic acid ammonium salt (eqn. 2): ##STR2##

A novel microbe Acidovorax facilis 72 W (ATCC 55746) has been isolatedfrom soil samples which had been exposed to aliphatic nitrites ordinitriles, and which could utilize 2-ethylsuccinonitrile as a nitrogensource. When used as a microbial whole-cell catalyst for the hydrolysisof unsymmetrically substituted α-alkyl-α,ω-dinitriles such as2-methylglutaronitrile (2-MGN) or 2-ethylsuccinonitrile (2-ESN), amixture of products is obtained. Over the course of the hydrolysisreactions, the corresponding dicarboxylic acid monoamides anddicarboxylic acids are produced in addition to the desiredω-nitrilecarboxylic acid. It was discovered that heating a suspension ofAcidovorax facilis 72 W (ATCC 55746) in a suitable buffer at 50° C. fora short period of time deactivates an undesirable nitrile hydrataseactivity of the whole-cell catalyst which catalyzed the production ofthe undesirable dicarboxylic acid monoamides (and which were furtherconverted by an amidase to the corresponding dicarboxylic acid). In thismanner, a whole-cell catalyst is prepared which contains a nitrilaseactivity which converts an α-alkyl-α,ω-dinitrile to only theω-nitrilecarboxylic acid ammonium salt resulting from hydrolysis of theω-nitrile group.

Heat-treatment of suspensions of Acidovorac facilis 72 W (ATCC 55746) at50° C. for one hour produces a microbial whole-cell catalyst whichhydrolyzes 2-methylglutaronitrile (2-MGN) to 4-cyanopentanoic acid(4-CPA) ammonium salt, 2-methyleneglutaronitrile (2-MEGN) to4-cyano-4-pentenoic acid (4-CPEA) ammonium salt, or2-ethylsuccinonitrile (2-ESN) to 3-cyanopentanoic acid (3-CPA) ammoniumsalt with extremely high regioselectivity, such that at completeconversion of the dinitrile, at least a 98% yield of theω-nitrilecarboxylic acid ammonium salt is produced by hydrolysis of theω-nitrile group (Table 1):

                  TABLE 1    ______________________________________              w-nitrile/acid                            concentration                                       yield    α,ω-dinitrile              ammonium salt (M)        (%)    ______________________________________    2-MGN     4-CPA(NH.sub.4.sup.+)                            0.10       99.3    "         "             0.40       99.4    "         "             1.00       98.7    "         "             1.85       100    "         "             2.00       100    2-MEGN    4-CPEA(NH.sub.4.sup.+)                            1.25       100    "         "             2.00       100    2-ESN     3-CPA(NH.sub.4.sup.+)                            0.10       100    "         "             0.40       100    "         "             1.00       100    "         "             1.25       100    ______________________________________

There are currently no non-enzymatic methods for the selectivehydrolysis of only one nitrile group of an aliphatic dinitrile to eitheran amide group or a carboxylic acid group at complete conversion of thedinitrile. If such a reaction is run to incomplete conversion (<20%conversion) in order to obtain a high selectivity to a monoamide ormonoacid hydrolysis product, a separation step is then required toisolate the product from unreacted dinitrile, and for recycle ofdinitrile into a subsequent reaction. Non-enzymatic hydrolysis reactionsalso typically involve heating solutions of the nitrile or dinitrile atelevated temperatures, often times in the presence of strong acid orbase, while the enzyme-catalyzed reaction described above are carriedout at ambient temperature in aqueous solution and at neutral pH with noadded acid or base.

Two mutants of the Acidovorax facilis 72 W (ATCC 55746) strain have beenprepared which produce only very low levels of the undesirable nitrilehydratase activity responsible for the formation of undesirablebyproducts. These mutant strains, Acidovorax facilis 72-PF-15 (ATCC55747) and Acidovorax facilis 72-PF-17 (ATCC 55745). do not requireheat-treatment ot the cells prior to use as catalyst for the hydrolysisof an aliphatic (α,ω-dinitrile to the corresponding ammonium salt of aω-nitrilecarboxylic acid. A comparison of the yields of 4-CPA and2-methylglutaric acid (2-MGA) produced by the hydrolysis of 2-MGN usinguntreated and heat-treated Acidovora facilis 72 W (ATCC 55746), and theuntreated Acidovora facilis 72-PF-15 (ATCC 55747) mutant strain areshown in Table 2:

                  TABLE 2    ______________________________________                       2-MGN!  4-CPA    2-MGA    catalyst          (M)      (% yield)                                        (% yield)    ______________________________________    A. facilis 72W, untreated                      0.10     62.7     34.6    A. facilis 72W, heat-treated                      0.10     99.3     0.7    A. facilis 72-PF-15, untreated                      0.10     96.8     3.6    A. facilis 72W, heat-treated                      0.40     99.4     0.6    A. facilis 72-PF-15, untreated                      0.40     98.8     1.2    A. facilis 72W, heat-treated                      1.00     98.7     1.3    A. facilis 72-PF-15, untreated                      1.00     99.2     0.8    ______________________________________

When heat-treated Acidovorax facilis 72 W (ATCC 55746) is used as acatalyst for the hydrolysis of aqueous solutions of the unsubstitutedaliphatic α,ω-dinitriles succinonitrile (SCN, 1.25M) or glutaronitrile(GLN, 1.5M), the corresponding ω-nitrilecarboxylic acid ammonium salts3-cyanopropionic acid (3-CPRA) and 4-cyanobutyric acid (4-CBA) areproduced in yields of 99.7% and 92.3%, respectively, and thecorresponding dicarboxylic acids are the only observed byproducts. Whenthis same catalyst is used to convert adiponitrile (ADN) to5-cyanopentanoic acid (5-CPA) ammonium salt, adipic acid (ADA)diammonium salt is the major product (>50% yield). Neither Acidovoraxfacilis 72 W (ATCC 55746) nor Acidovorax facilis 72-PF-15 (ATCC 55747)are suitable as catalyst for the preparation of 5-CPA ammonium salt inhigh yield.

More than 30 different microbial cultures isolated from soil sampleswhich had been exposed to aliphatic nitriles or dinitriles, and whichcould grow on various nitriles or amides as nitrogen source, werescreened for high selectivity for 5-CPA production. A second novelmicrobe, Comamonas testosteroni 5-MGAM-4 D (ATCC 55744), was isolated(using 2-methylglutaramide as nitrogen source) which contained severalnitrile hydratase and amidase activities. When used as a whole cellcatalyst for the hydrolysis of ADN, the resulting product mixture iscomposed primarily of ADA diammonium salt, adipamide (ADAM) and adipamicacid (ADMA), with only a minor yield of 5-CPA ammonium salt observed. Itwas again tound that heating the microbe at 50° C. for a short period oftime deactivated an undesirable nitrite hydratase activity to a greatextent, leaving the microbial catalyst with a nitrite hydratase activitywhich converts ADN to 5-cyanovaleramide, and an amidase which converts5-cyanovaleramide to 5-CPA ammonium salt. Heat-treatment of Comamonastestosteroni 5-MGAM-4 D at 50° C. for one hour results in a microbialcell catalyst which produces 5-CPA in yields as high as 97% at completeconversion of ADN.

The ability to eliminate unwanted nitrile hydratase by heat treatment,while at the same time leaving a relatively heat-stable nitrilaseactivity or nitrite hydratase activity for the conversion of dinitrilesto the cyano acids was not previously known and could not have beenpredicted because the temperature stability of the nitrilase or nitritehydratase enzyme was unknown. It is expected that heat treatment attemperatures between 35° C. and 70° C. for between 10 and 120 minuteswill produce the described useful effect.

B. Preparation of five- or six-membered ring lactams.

A method has been discovered for the preparation of five-membered ringor six-membered ring lactams in high yields by the direct hydrogenationof the ω-nitrilecarboxylic acid ammonium salt product mixture producedby the enzyme-catalyzed hydrolysis of aliphatic α,ω-dinitriles inaqueous solution. This method does not require the isolation of theω-nitrilecarboxylic acid ammonium salt from the product mixture of thehydrolysis reaction prior to the hydrogenation step, nor does it requirethe conversion of the ω-nitrilecarboxylic acid ammonium salt to the freeacid (e.g., conversion of 4-CPA ammonium salt to 4-CPA) prior tohydrogenation, or isolation of the resulting ω-aminocarboxylic acidammonium salt from the hydrogenation product mixture and conversion ofthe ammonium salt to the free carboxylic acid prior to cyclization.

After producing an aqueous product mixture containing the ammonium saltof a ω-nitrilecarboxylic acid from an aliphatic α,ω-dinitrile by usingan enzyme catalyst (eqn. 3), removal of the enzyme catalyst and reactionof the resulting aqueous solution with hydrogen and a stoichiometricexcess of added ammonia (as ammonium hydroxide) in the presence of asuitable hydrogenation catalyst was expected to produce an aqueoussolution containing an aliphatic ω-aminocarboxylic acid ammonium salt(eqn. 4): ##STR3## The use of an excess of ammonia during thehydrogenation of a nitrile to the corresponding amine is necessary tolimit reductive alkylation of an imine intermediate (produced during thehydrogenation of the nitrile group to an amine) by the productω-aminocarboxylic acid, which results in dimer formation and yield loss.This technique is well-documented (De Bellefon et al., Catal. Rev. Sci.Eng., (1994), vol. 36, 459.506) and is commonly practiced by thoseskilled in the art of hydrogenation of nitrites.

Based on the prior art, it was expected that the ω-aminocarboxylic acidammonium salt produced by the hydrogenation of a ω-nitrilecarboxylicacid ammonium salt would have to be converted to the free acid andisolated (eqn. 5) before a thermally-induced cyclization reaction toproduce the desired lactam could be performed (eqn. 6). According toMares et al. (supra), 6-aminocaproic acid (6-ACA) (eqn. 6, n=3, R=H) andcaprolactam exist as a reversible equilibrium mixture at concentrationsof less than 1.0 mol/kg (ca. 1.0M) in water and the concentration ofcaprolactam increases with increasing temperature. At a totalconcentration of 6-ACA and caprolactam of 0.85 mol/kg (ca. 0.85M), thepercentage of caprolactam was reported by Mares et al. to increase from38.7% at 180° C. to 92.2% at 250° C. ##STR4##

In the present case, where an excess of added ammonia (as ammoniumhydroxide) is present and the pH of the reaction mixture is between pH 9and pH 10, it was not expected that the ammonium salt of 6-ACA wouldcyclize to produce significant amounts of caprolactam at hydrogenationtemperatures of less than 200° C. The pKa's of the carboxylic acid groupand the protonated amine group of 6-aminocaproic acid are 4.373 and10.804, respectively (Lange's Handbook of Chemistry, J. A. Dean, ed.,14th edn., (1992), McGraw-Hill, N.Y., p. 8.222 (as 6-aminohexanoicacid)), and the pKa of NH₄ ⁺ is 9.25. At a reaction mixture pH ofbetween 9 and 10, it can be calculated that at least 99.997% of the6-ACA exists in solution as the ammonium salt of the carboxylic acid,and additionally, approximately half of the amine groups of the6-aminocaproic acid ammonium salt are also protonated. Therefore, it wasnot expected that significant amounts of the 6-ACA ammonium salt wouldcyclize to produce caprolactam under the hydrogenation conditions of thepresent invention, where displacement of a hydroxyl anion (⁻ OH) fromthe cyclic reaction intermediate is not possible (eqn. 7): ##STR5##

When hydrogenations of aqueous solutions of 5-CPA ammonium salt(prepared by enzymatic hydrolysis of ADN) are performed in the presenceof from 0M to 2.0M NH₄ OH at temperatures of from 70° C. to 160° C.,complete conversion of 5-CPA to 6-ACA ammonium salt was observed withlittle byproduct formation, and, as predicted, less than 3% yields ofcaprolactam (the resulting seven-membered ring lactam) are obtained(Table 3):

                  TABLE 3    ______________________________________    Temp. 5-CPA ammo-  NH.sub.4 OH!                               time 5-CPA  caprolactam    (°C.)          nium salt (M)                      (M)      (h)  conv. (%)                                           (%)    ______________________________________    70    1.0         0        2    100     0.7    70    1.0         1.0      2    100     0.7    70    1.0         1.5      2    94      0.8    70    1.0         2.0      2    84      0.8    120   1.0         0        2    99      0.9    120   1.0         1.0      2    100     0.9    120   1.0         1.5      2    97      1.0    120   1.0         2.0      2    97      1.1    160   1.0         0        2    97      2.5    160   1.0         1.0      2    84      2.3    160   1.0         1.5      2    97      3.0    160   1.0         2.0      2    95      2.7    ______________________________________

Yields of caprolactam of less than 3% are also obtained when aqueous onsprepared by mixing authentic 6-ACA and ammonium hydroxide are treatedunder the same hydrogenation reaction conditions as for hydrogenation ofthe ammonium salt of 5-CPA prepared by enzymatic hydrolysis of ADN. Thecyclization of the ammonium salt of 6-ACA in aqueous solutions preparedby the hydrogenation of 5-CPA ammonium salt at 70° C. was attempted attemperatures greater than 200° C. by first removing the hydrogenationcatalyst, then heating the ca. 1.0M 6-ACA ammonium salt product mixturesat 280° C. for 2 h. Yields of caprolactam of less than 18% were produced(Table 4). These results demonstrate that only very low yields ofcaprolactam are obtained by the direct hydrogenation of aqueoussolutions of 5-CPA ammonium salt in the presence of excess ammonia. Thiswas expected in light of the predicted inability of the 6-ACA ammoniumsalt to undergo cyclization, particularly in the presence of excessammonium hydroxide.

                  TABLE 4    ______________________________________    Temp.  6-ACA ammonium salt                           NH.sub.4 OH!                                   time  caprolactam    (°C.)           (M)            (M)      (h)   (%)    ______________________________________    280    1.0            0        2     17.8    280    1.0            1.0      2     17.2    280    1.0            1.5      2     11.0    280    1.0            2.0      2     9.0    280    1.0            2.5      2     3.1    ______________________________________

It was also expected that when aqueous mixtures of 3-cyanopentanoic acid(3-CPA) ammonium salt (prepared by enzymatic hydrolysis of2-ethylsuccinonitrile (2-ESN)) or 4-cyanovaleric acid (4-CPA) ammoniumsalt (prepared by enzymatic hydrolysis of 2-methylglutaronitrile(2-MGN)) were hydrogenated at from 70° C. to 160° C. in the presence ofexcess ammonia, the corresponding ω-aminocarboxylic acid ammonium saltswould be produced, and that these salts would have to be isolated as thefree acids before the cyclization reaction to the corresponding lactamcould be performed (as was the case for 5-CPA ammonium salt). Althoughthe pKa's of the carboxylic acid and the protonated aminefunctionalities of the product 4-amino-3-ethylbutyric acid or5-amino-4-methylpentanoic acid have not been reported, it is reasonableto assume that these pKas are similar to those of the 6-ACA isomer.

Unexpectedly, hydrogenation of aqueous solutions of 3-CPA ammonium salt(produced by enzymatic hydrolysis of 2-ESN) in the presence of Raneynickel and excess ammonia at pH 9-10 and at temperatures of from 70° C.to 180° C. for 2 h produce the corresponding lactam4-ethylpyrrolidin-2-one (4-EPRD) directly, and at yields of up to 91%(Table 5). The yield of 4-EPRD also increases with increasingconcentration of added ammonium hydroxide (added in addition to theammonium ion concentration already present as the ammonium salt of thecarboxylic acid), which indicates the desirability of performing thehydrogenation of aqueous solutions of the ammonium salts of themononitrile acids in the presence of added ammonia in order to limitwell-known reductive alkylation reactions which produce dimer andpolymer (Table 6).

                                      TABLE 5    __________________________________________________________________________    Temp.        3-CPA ammonium salt                   NH.sub.4 OH!                       wt. %                            time                               3-CPA                                    4-EPRD    (°C.)        (M)       (M)  Raney Ni                            (h)                               (% conv.)                                    (% yield)    __________________________________________________________________________    70  1.0       2.0  5    2  7    1.5    120 1.0       2.0  5    2  55   22.7    140 1.0       2.0  5    2  71   55.4    140 1.0       2.0  10   2  100  89.9    160 1.0       2.0  5    2  100  90.1    160 1.0       2.0  10   2  100  91.3    180 1.0       2.0  5    2  100  86.1    180 1.0       2.0  10   2  100  90.0    __________________________________________________________________________

                                      TABLE 6    __________________________________________________________________________    Temp.        3-CPA ammonium salt                   NH.sub.4 OH!                       wt. %                            time                               3-CPA                                    4-EPRD    (°C.)        (M)       (M)  Raney Ni                            (h)                               (% conv.)                                    (% yield)    __________________________________________________________________________    160 1.0       0    5    2  99   80.1    160 1.0       1.0  5    2  99   87.6    160 1.0       2.0  5    2  100  90.1    160 1.0       3.0  5    2  100  85.4    180 1.0       0    5    2  100  75.1    180 1.0       1.0  5    2  100  85.8    180 1.0       2.0  5    2  100  88.5    180 1.0       3.0  5    2  100  90.0    __________________________________________________________________________

The hydrogenation of aqueous solutions of 4-CPA ammonium salt (producedby enzymatic hydrolysis of 2-MGN) in the presence of Raney nickel andexcess ammonia at pH 9-10 and at 160° C. for 2 h produces thecorresponding lactam 5-methyl-2-piperidone (5-MPPD) directly, and atyields as high as 96% (Table 7):

                                      TABLE 7    __________________________________________________________________________    Temp.        4-CPA ammonium salt                   NH.sub.4 OH!                       wt. %                            time                               4-CPA                                    5-MPPD    (°C.)        (M)       (M)  Raney Ni                            (h)                               (% conv.)                                    (% yield)    __________________________________________________________________________    160 1.0       0    5    2  100  85.6    160 1.0       2.0  5    3  100  96.4    180 1.0       2.0  5    2  100  91.4    180 1.0       3.0  5    2  100  89.5    __________________________________________________________________________

Hydrogenation of aqueous solutions of the ammonium salts of3-cyanopropionic acid (3-CPRA) or 4-cyanobutyric acid (4-CBA) (producedby enzymatic hydrolysis of the corresponding α,ω-dinitriles) produce thecorresponding lactams 2-pyrrolidinone and 2-piperidone in 91.0% yieldand 93.5% yield, respectively. Hydrogenation of aqueous solutions of4-cyano4-pentenoic acid (4-CPEA) ammonium salt (produced by enzymatichydrolysis of 2-methyleneglutaronitrile) result in hydrogenation of boththe nitrile and carbon-carbon double bond to produce5-methyl-2-piperidone in up to 85% yield.

By adding an excess of ammonia (as ammonium hydroxide) to thehydrogenation reactions in order to limit reductive alkylation duringthe hydrogenation of nitrites to amines, several additionalbyproduct-forming reactions could also have occurred (De Bellefon etal., Catal. Rev. Sci. Eng., (1994), vol. 36, 459.506). It is well-knownto those skilled in the art that a common method for the preparation ofan amide or carboxylic acid from a nitrile is to heat an aqueous mixtureof the nitrile in the presence of an acid or base catalyst. Therefore,an expected competing reaction of the ammonium salt of a mononitrilecarboxylic acid under the hydrogenation conditions used in the presentinvention would be the base-catalyzed hydrolysis of the nitrile group toproduce either the dicarboxylic acid monoamide ammonium salt or thedicarboxylic acid diammonium salt. These unwanted amide/acid anddicarboxylic acid ammonium salts byproducts are produced during thehydrogenations, but in very low yields compared to the yields oflactarn.

A second byproduct-forming reaction between the excess ammonia presentand the product lactam could have produced an equilibrium mixture of thelactam with the expected ammonolysis product, an ω-aminocarboxamide. Thehigh yields of the five-membered and six-membered ring lactams attainedunder the present reaction conditions suggest that the ammonolysis (orbase-catalyzed hydrolysis) of the product lactams is not significant.

In addition to producing lactams from aliphatic α,ω-dinitriles,N-methyl-lactams are prepared by the substitution of methylamine forammonia in the hydrogenation of aqueous solutions of the ammonium saltsof 4-CPA or 3-CPA. Addition of from one to four equivalents ofmethylamine (pKa 10.62 for the protonated amine) to an aqueous solutionof 4-CPA containing one equivalent of ammonium ion (pKa 9.25) wasexpected to produce a significant amount of free ammonia (due to therelative differences in pKa's of protonated methylamine and ammoniumions in water). This free ammonia could then compete with unprotonatedmethylamine for reaction with the intermediate imine produced during thehydrogenation of the nitrile group, leading to the production of amixture of the ammonium salts of 5-amino-4-methylpentanoic acid and5-N-methylamino-4-methylpentanoic acid, respectively, which in turncyclize to produce 5-MPPD and 1,5-dimethyl-2-piperidone (1,5-DMPD),respectively.

The relative yields of 5-MPPD and 1,5-DMPD produced by hydrogenation of1.0M aqueous solutions of the ammonium salt of 4-CPA are found to bedependent on the choice of catalyst. Raney nickel and ruthenium onalumina each produce 5-MPPD as the major lactam product, even in thepresence of 3.0M methylamine, while 1,5-DMPD is the major product whenusing 5% Pd/C or 4.5% Pd/0.5% Pt/C as catalyst at the same methylamineconcentration. When using 5% Pd/C as catalyst, the yield of 1,5-DMPDincreases with increasing concentration of methylamine (Table 8).

The substitution of 2.0M methylamine for ammonia in the hydrogenation ofan aqueous solutions of 1.0M 3-CPA ammonium salt at 140° C. and using aPd/C catalyst produces 4-ethyl-1-methylpyrrolidin-2-one (4-EMPRD) and4-EPRD in 69.8% and 20.4% yields, respectively, at 96% conversion.

                                      TABLE 8    __________________________________________________________________________                                 1,5-    Temp.        4-CPA(NH.sub.4.sup.+)               CH.sub.3 NH.sub.2                         time                            4-CPA                                 DMPD 5-MPPD    (°C.)        (M)    (M)  catalyst                         (h)                            (% conv)                                 (% yield)                                      (% yield)    __________________________________________________________________________    160 1.0    3.0  Ra--Ni                         2  100  19.2 68.5    160 1.0    3.0  RuAl.sub.2 O.sub.3                         2  100  19.0 63.5    140 1.0    3.0  Pd/C 2  99   83.4 0    160 1.0    1.0  Pd/C 2  100  53.5 0    160 1.0    1.25 Pd/C 2  100  68.3 0    160 1.0    1.5  Pd/C 2  100  76.4 0    160 1.0    2.0  Pd/C 2  100  81.5 0    160 1.0    3.0  Pd/C 2  100  81.7 7.8    160 1.0    4.0  Pd/C 2  100  88.4 1.9    180 1.0    3.0  Pd/C 2  97   73.0 6.6    160 1.4    2.3  Pd/Pt/C                         2  99   94.0 3.1    __________________________________________________________________________

DESCRIPTION OF THE PREFERRED EMBODIMENTS Methods and Materials

Microbial Catalysts for the Preparation of ω-Nitrilecarboxylic Acids

Two microorganisms have been isolated for use as a microbial catalystfor the conversion of aliphatic α,ω-dinitriles to the correspondingω-nitrilecarboxylic acids: Acidovorax facilis 72 W (ATCC 55746) andComamonas testosteroni 5-MGAM-4 D (ATCC 55744).

Acidovorax facilis 72 W (ATCC 55746) was isolated from soil collected inOrange, Tex. Standard enrichment procedures were used with the followingmedium (E2 Basal Medium, pH 7.2):

    ______________________________________    E2 Basal Medium        g/L    ______________________________________    KH.sub.2 PO.sub.4      1.4    NaH.sub.2 PO.sub.4     0.69    Sodium citrate         0.1    CaCl.sub.2.2H.sub.2 O  0.025    KCl                    0.5    NaCl                   1.0    MgSO.sub.4.7H.sub.2 O  0.5    FeSO.sub.4.7H.sub.2 O  0.05    CoCl.sub.2.6H.sub.2 O  0.01    MnCl.sub.2.4H.sub.2 O  0.001    ZnCl.sub.2             0.0005    NaMoO.sub.4.2H.sub.2 O 0.0025    NiCl.sub.2.6H.sub.2 O  0.01    CuSO.sub.4.2H.sub.2 O  0.005    Biotin                 0.0002    Folic Acid             0.0002    Pyridoxine.HCl         0.001    Riboflavine            0.0005    Thiamine.HCl           0.00005    Nicotinic Acid         0.0005    Pantothenic Acid       0.0005    Vitamin B.sub.12       0.00001    p-Aminobenzoic Acid    0.0005    ______________________________________

The following supplementations were made to the E2 basal medium for theenrichments described above:

    ______________________________________    Strain    Enrichment Nitrogen Source                               Other Supplements    ______________________________________    A. facilis 72W              0.2% (v/v) ethylsuccinonitrile                               0.3% (v/v) glycerol    ______________________________________

Strains were originally selected based on growth and ammonia productionon the enrichment nitrile. Isolates were purified by repeated passing onBacto® Brain Heart Infusion Agar (Difco, Detroit, Mich.) followed byscreening for ammonia production from the enrichment nitrile. Purifiedstrains were identified based on their carbon source utilization profileon a Biolog® test system (Hayward, Calif., USA) using Gram negative testplates.

For testing nitrile hydrolysis activity, E2 basal medium with 10 g/Lglucose was used to grow A. facilis 72 W. The medium was supplementedwith 25 mM (±)-2-methylglutaronitrile. A 10 mL volume of supplemented E2medium was inoculated with 0.1 mL of frozen stock culture. Followingovernight growth at room temperature (22-25° C.) on a shaker at 250 rpm,the 10 mL inoculum was added to 990 mL of fresh medium in a 2 L flask.The cells were grown overnight at room temperature with stirring at arate high enough to cause bubble formation in the medium. Cells wereharvested by centrifugation, washed once with 50 mM phosphate buffer(pH7.2)/15% glycerol and the concentrated cell paste was immediately frozenon dry ice and stored at -65° C. Adiponitrile, 10 mM, was also used inthe 1 liter fermentations. Fermentations were stopped after 16-20 hoursof growth. The cell suspension was chilled to 4° C., harvested bycentrifugation and frozen at -60° C. following one wash with 15%glycerol in 0.05M phosphate buffer, pH 7.2. Thawed cell pastes were usedfor testing nitrile hydrolysis activity. The desired property of themicroorganism is a nitrile hydrolyzing activity capable of regiospecificattack of a dinitrile compound in the absence of interfering amidaseactivity. Microorganisms tend to undergo mutation. Some mutations may befavorable to the desired nitrile conversion. Thus, even mutants of thenative strain may be used to carry out the process of the instantinvention.

Standard enrichment procedures were also used for Comamonas testosteroni5-MGAM-4 D with E2 Basal Medium, pH 7.2 modified by having vitamins atone tenth the concentration in the standard basal medium describedabove. The following supplementations were made to the modified E2 basalmedium for the enrichments:

    ______________________________________    Strain    Enrichment Nitrogen Source                               Other Supplements    ______________________________________    C. testosteroni              2-Methylglutamide                               glycerol (0.6%)    5-MGAM-4D (MGAM; 1.0% w/v)    ______________________________________

Strains were originally selected based on growth on the enrichmentnitrileamide. Isolates were purified by repeated passing on agar platesusing the above medium. Purified strains were identified based on theircarbon source utilization profile on a Biolog® test system (Hayward,Calif., USA) using Gram negative test plates.

For testing nitrile hydrolysis activity, modified E2 basal medium with6.0 g/L of either glucose or glycerol was used to grow cell material.The medium was supplemented with 1.0% MGAM. A 250 mL unbaffled shakeflask containing 50 mL of supplemented E2 medium was inoculated with 0.2mL of frozen stock culture and grown for 72 h at 30° C. on a shaker at200 rpm. The cells were harvested by centrifugation and washed with 10mL of 20 mM KH2PO4, pH 7.0. The cells were screened in 10 mL reactionscontaining 20 mM KH2PO4, pH 7.0 and 0.1M of either methylglutaronitrileor methylglutaramide for regiospecific hydrolysis using HPLC. Thedesired property of the microorganism is a nitrile hydrolyzing activitycapable of regiospecific attack of a dinitrile compound in the absenceof interfering amidase activity. Microorganisms tend to undergomutation. Some mutations may be favorable to the desired nitriteconversion. Thus, even mutants of the native strain may be used to carryout the process of the instant invention.

The present invention is not limited to the particular organismsmentioned above, but includes the use of variants and mutants thereofthat retain the desired property. Such variants and mutants can beproduced from parent strains by various known means such as x-rayradiation, UV-radiation, and chemical mutagens.

To produce biocatalyst for process demonstration (Examples 1-26), thefollowing media were used.

    ______________________________________    Strain   Medium    ______________________________________    72-PF-15 Lauria-Bertani Medium(Bacto ® tryptone,             10 g/L + Bacto ® yeast             extract, 5 g/L + NaCl, 10 g/L) + 0.5%(w/v) sodium             succinate.6H.sub.2 O    72 W     E2 + 1% (w/v) glucose + 0.4% (w/v) adipamide    5-MGAM-4D             E2 + 1% (w/v) glucose + 0.2% (w/v) propionamide    ______________________________________

To initiate growth, 10 mL of the appropriate medium was inoculated with0.1 mL of frozen stock culture. Following overnight growth at 28° C.with shaking at 250 rpm, the growing cell suspension was transferred to1 L of the same medium in a 2 L flask and growth continued at 28° C.with shaking. The 1 L growing cell suspension was then added to 9 L ofthe same medium in a 10 L fermentation vessel where growth continued.Nominal conditions in the fermenter were: ≧80% oxygen saturation, 25°C., pH 7.2, 300-1000 rpm. After 20-91 hours, the vessel was chilled to8-12° C. and glycerol added to 10% final concentration. Cell materialwas harvested by centrifugation. The concentrated cell paste wasimmediately frozen on dry ice and stored at -70° C. until use. Numerousother supplementations which will serve as carbon and nitrogen sourcesfor cell growth in E2 basal medium are known to those skilled in theart. These, as well as complex nutrient media, can be used to producebiocatalyst. The particular media described above should not be viewedas restrictive.

Selection of Mutant Strains of Acidovorax facilis 72 W Deficient inNHase Activity.

Mutants of Acidovorax facilis 72 W (ATCC 55746) with reduced capacity toproduce the undesirable 2-methylglutaric acid by-product duringhydrolysis of 2-MGN to 4-CPA were selected based on their inability touse 2-MGN as a carbon and energy source. Specifically, an overnightculture of strain A. facilis 72 W grown on LB/succinate medium (1% (w/v)Bacto-tryptone (Difco, Detroit, Mich., USA), 0.5% (w/v) Bacto-yeastextract (Difco), 1% (w/v) NaCl, 0.5% (w/v) sodium succinate hexahydrate)was exposed to 100 μg/mL solution ofN-methyl-N'-nitro-N-nitrosoguanidine, a mutagenic agent, forapproximately 30 minutes. This resulted in a 99.9% reduction in viablecells in the culture. Mutagenized cells were washed free of the mutagenby centrifugation in sterile, 1M sodium phosphate buffer, pH 7.2. Washedcells were resuspended in LB/succinate medium and grown overnight at 30°C. Cells were then washed by centrifugation in sterile, 50 mM sodiumphosphate buffer, pH 7.2, and resuspended in E2 minimal medium (withoutglucose) containing 0.2% (v/v) 2-methylglutaronitrile, and theantibiotics cycloserine, 0.2 mg/mL and piperacillin, 40 μg/mL. Cellswere incubated overnight at 30° C. and again washed in sterile, 50 mMsodium phosphate buffer, pH 7.2. Washed cells were spread on agar platescontaining a non-selective medium: E2 minimal medium(without glucose)plus 0.2% (v/v) 2-methylglutaronitrile and 0.5% (w/v) sodium succinatehexahydrate, at a concentration of 40-100 colony-forming units perplate. Plates were incubated for approximately 48 h at 30° C. to allowcolonies to develop. Colonies which developed were replica plated ontoagar plates containing selective medium: E2 minimal medium(withoutglucose) plus 0.2% (v/v) 2-MGN. Plates were incubated 48 h at 30° C. toallow colonies to develop. Mutants with desirable qualities do not growwell on the selective medium. Therefore, after 48 h, replicated plateswere compared and strains showing growth only on non-selective mediumwere saved for further testing.

In total, approximately 5,120 colonies were checked from 89 plates and19 strains with the desirable qualities were identified. These mutantstrains were further tested for growth in liquid, E2 minimalmedium(without glucose) plus 0.2% (v/v) 2-MGN. Strains which showedlittle or no growth in this medium were screened for their ability toproduce 2-methylglutaric acid during growth in liquid medium consistingof E2 minimal medium (without glucose) plus 0.2% v/v) 2-MGN and 0.5%(w/v) sodium succinate hexahydrate. As a result of this process. twomutant strains, identified as Acidovorax facilis 72-PF-15 (ATCC 55747)and Acidovurax facilis 72-PF17 (ATCC 55745) were chosen for furtherdevelopment due to their greatly diminished capacity to produce2-methylglutaric acid.

Aliphatic α,ω-Dinitrile Hydrolysis Reactions

An aqueous solution containing the ammonium salt of an aliphaticω-nitrilecarboxylic acid is prepared by mixing the correspondingaliphatic α,ω-dinitrile with an aqueous suspension of the appropriateenzyme catalyst (as identified in part A above). Whole microbial cellscan be used as catalyst without any pretreatment. Alternatively, theycan be immobilized in a polymer matrix (e.g., alginate beads orpolyacrylamide gel (PAG) particles) or on an insoluble solid support(e.g., celite) to facilitate recovery and reuse of the catalyst. Methodsfor the immobilization of cells in a polymer matrix or on an insolublesolid support have been widely reported and are well-known to thoseskilled-in-the-art. The nitrilase enzyme, or nitrile hydratase andamidase enzymes, can also be isolated from the whole cells and useddirectly as catalyst, or the enzyme(s) can be immobilized in a polymermatrix or on an insoluble support. These methods have also been widelyreported and are well-known to those skilled in the art.

Some of the aliphatic α,ω-dinitriles used as starting material in thepresent invention are only moderately water soluble. Their solubility isalso dependent on the temperature of the solution and the saltconcentration (buffer and/or ω-nitrilecarboxylic acid ammonium salt) inthe aqueous phase. For example, adiponitrile was determined to have asolubility limit of ca. 0.60M, (25° C., 20 mM phosphate buffer, pH 7)and under the same conditions, 2-methylglutaronitrile was determined tohave a solubility limit of ca. 0.52M. In this case, production of anaqueous solution of a ω-nitrilecarboxylic acid ammonium salt at aconcentration greater than the solubilty limit of the startingα,ω-dinitrile is accomplished using a reaction mixture which isinitially composed of two phases: an aqueous phase containing the enzymecatalyst and dissolved α,ω-dinitrile, and an organic phase (theundissolved α,ω-dinitrile). As the reaction progresses, the dinitriledissolves into the aqueous phase, and eventually a single phase productmixture is obtained.

The concentration of enzyme catalyst in the reaction mixture isdependent on the specific catalytic activity of the enzyme catalyst andis chosen to obtain the desired rate of reaction. The wet cell weight ofthe microbial cells used as catalyst in hydrolysis reactions typicallyranges from 0.001 grams to 0.100 grams of wet cells per mL of totalreaction volume, preferably from 0.002 grams to 0.050 grams of wet cellsper mL. The specific activity of the microbial cells (IU/gram wet cellwt.) is determined by measuring the rate ot conversion ot a 0.10Msolution of a dinitrile substrate to the desired ω-nitrilecarboxyvicacid product at 25° C. using a known weight of microbial cell catalyst.An IU (International Unit) of enzyme activity is defined as the amountof enzyme activity required to convert one micromole of substrate toproduct per minute.

The temperature of the hydrolysis reaction is chosen to both optimizeboth the reaction rate and the stability of the enzyme catalystactivity. The temperature of the reaction may range from just above thefreezing point of the suspension (ca. 0° C.) to 60° C., with a preferredrange of reaction temperature of from 5° C. to 35° C. The microbial cellcatalyst suspension may be prepared by suspending the cells in distilledwater, or in a aqueous solution of a buffer which will maintain theinitial pH of the reaction between 5.0 and 10.0, preferably between 6.0and 8.0. As the reaction proceeds, the pH of the reaction mixture maychange due to the formation of an ammonium salt of the carboxylic acidfrom the corresponding nitrile functionality of the dinitrile. Thereaction can be run to complete conversion of dinitrile with no pHcontrol, or a suitable acid or base can be added over the course of thereaction to maintain the desired pH.

The final concentration of aliphatic ω-nitrilecarboxylic acid ammoniumsalt in the product mixture at complete conversion of the α,ω-dinitrilemay range from 0.001M to the solubility limit of the aliphaticω-nitrilecarboxylic acid ammonium salt. Typically, the concentration ofthe ω-nitrilecarboxylic acid ammonium salt ranged from 0.10M to 2.0M.The product mixture of the hydrolysis reaction may be used directly inthe subsequent hydrogenation reaction after recovery of the enzymecatalyst by centrifugation and/or filtration. The ω-nitrilecarboxylicacid may also be isolated from the product mixture (after removal of thecatalyst) by adjusting the pH of the reaction mixture to between 2.0 and2.5 with conc. HCl, saturation of the resulting solution with sodiumchloride, and extraction of the ω-nitrilecarboxylic acid with a suitableorganic solvent such as ethyl acetate, ethyl ether, or dichloromethane.The combined organic extracts are then combined, stirred with a suitabledrying agent (e.g., magnesium sulfate), filtered, and the solventremoved (e.g., by rotary evaporation) to produce the desired product inhigh yield and in high purity (typically 98-99% pure). If desired, theproduct can be further purified by recrystallization or distillation.

Hydrogenation/Cyclization of ω-Nitrilecarboxylic Acid Ammonium Salts

Catalytic hydrogenation is a preferred method for preparing an aliphaticamine from an aliphatic nitrite. In the present invention, theω-aminocarboxylic acid produced during the hydrogenation cyclizes to thecorresponding five-membered or six-membered ring lactam. An aqueoussolution of an ammonium salt of an ω-nitrilecarboxylic acid (prepared bycentrifugation and filtration of the aqueous product mixture produced bythe enzymatic hydrolysis of the corresponding aliphatic α,ω-dinitrile)is first mixed with concentrated ammonium hydroxide and water to producea solution which contains from one to four stoichiometric equivalents ofadded ammonium hydroxide. The ammonium hydroxide is added to limit thereductive alkylation of the ω-nitrilecarboxylic acid by the productω-aminocarboxylic acid during the course of the hydrogenation. A two- tothree-fold stoichiometric excess of the ammonium hydroxide relative tothe amount of ω-nitrilecarboxylic acid present in the reaction mixtureis preferred to achieve an optimum yield of the desired lactam.Optionally, ammonia gas can be substituted for the ammonium hydroxideadded to the reaction mixture. The initial concentration of theω-nitrilecarboxylic acid ammonium salt in the hydrogenation reactionmixture may range from 5 weight percent to 20 weight percent of thesolution, with a preferred range of from 7.5 weight percent to 12.5weight percent.

For the preparation of a N-methyllactam, methylamine is substituted forammonium hydroxide or ammonia, using one to four stoichiometricequivalents relative to the amount of ω-nitrilecarboxylic acid presentin the reaction mixture. A three-fold to four-fold stoichiometric excessof methylamine relative to the amount of ω-nitrilecarboxylic acidpresent in the reaction mixture is preferred to achieve an optimum yieldof the desired N-methyllactam.

To the hydrogenation reaction mixture described above is then added asuitable hydrogenation catalyst, and the resulting mixture heated underpressure with hydrogen gas to convert the ω-nitrilecarboxylic acidammonium salt to the corresponding five-membered or six-membered ringlactam. Hydrogenation catalysts suitable for this purpose include (butare not limited to) the various platinum metals, such as iridium,osmium, rhodium, ruthenium, platinum, and palladium; also various othertransition metals such as cobalt, copper, nickel and zinc. The catalystmay be unsupported, (for example as Raney nickel or platinum oxide), orit may be supported (for example, as palladium on carbon, platinum onalumina, or nickel on kieselguhr).

The hydrogenation catalyst is used at a minimum concentration sufficientto obtain the desired reaction rate and total conversion of startingmaterials under the chosen reaction conditions. This concentration iseasily determined by trial. The catalyst may be used in amounts of from0.001 to 20 or more parts by weight of catalyst per 100 parts ofω-nitrilecarboxylic acid employed in the reaction. The catalyst loadingin the reaction mixture is typically from 1% to 10% (weightcatalyst/weight of ω-nitrilecarboxylic acid), with a 3% to 5% catalystloading preferred. Raney nickel (e.g., Cr-promoted Raney nickel catalyst(Grace Davison Raney 2400 active metal catalyst)) is a preferredcatalyst for reactions run in the presence of added ammonia to producelactams. while 5% or 10% palladium on carbon. or 4.5% palladium/0.5%platinum on carbon are preferred for reactions run in the presence ofadded methylamine to produce N-methyllactams.

The hydrogenation temperature and pressure can vary widely. Thetemperature may generally be in the range of from 45° C. to 200° C.,preferably from 70° C. to 180° C. The hydrogen pressure is generally inthe range of from about atmospheric to about 100 atmospheres, preferablyfrom 30 to 60 atmospheres. The hydrogenation is performed without any pHadjustment of the reaction mixture, which with the addition of an excessof ammonium hydroxide, ammonia, or methylamine is generally between a pHof from 9 to 12. Within this pH range, the exact value may be adjustedto obtain the desired pH by adding any compatible, non-interfering baseor acid. Suitable bases include, but are not limited to, alkali metalhydroxides, carbonates, bicarbonates and phosphates, while suitableacids include, but are not limited to, hydrochloric, sulfric, orphosphoric acid.

Lysis of the microbial cell catalysts during the hydrolysis reaction orcontaminants present from the catalyst preparation could have introducedcompounds into the hydrogenation reaction mixture (e.g., thiols) whichcould have poisoned the catalyst activity. No poisoning or deactivationof the hydrogenation catalysts was observed when comparing thehydrogenation of ω-nitrilecarboxylic acid ammonium salt mixturesproduced via microbial hydrolysis with the hydrogenation of aqueoussolutions of the same ω-nitrilecarboxylic acid which was isolated fromthe hydrolysis product mixtures and purified prior to hydrogenation.

The lactam or N-methyllactam may be readily isolated from thehydrogenation product mixture by first filtering the mixture to recoverthe hydrogenation catalyst, and then the product can be distilleddirectly from the resulting aqueous filtrate. The ammonia producedduring the cyclization reaction or added to the reaction mixture canalso be recovered for recycling by this distillation process, and thegeneration of undesirable inorganic salts as waste products is avoided.The lactams or N-methyl lactams may also be recovered by filtering thehydrogenation mixture, adjustment of the filtrate to a pH of ca. 7 withconc. HCl and saturation with sodium chloride, extraction of the lactarnor N-methyllactam (batch or continuous extraction) with an organicsolvent such as ethyl acetate, dichloromethane, or ethyl ether, andrecovery from the organic extract by distillation or crystallization. Inthe accompanying examples, the isolated yields reported for this methodare unoptimized, and this method was used simply to obtain purifiedproduct for analysis and confirmation of chemical identity.

In the following examples. which serve to further illustrate theinvention and not to limit it, the % recovery of aliphaticα,ω-dinitriles and the % yields of the hydrolysis products formed duringthe microbial hydrolysis reactions were based on the initial amount ofα,ω-dinitrile present in the reaction mixture (unless otherwise noted),and determined by HPLC using a refractive index detector and either aSupelcosil LC-18-DB column (25 cm×4.6 mm dia.) or a Bio-Rad HPX-87Hcolumn (30 cm×7.8 mm dia.). The yields of lactams and N-methyl-lactamsproduced by the hydrogenation of aqueous solutions ofω-nitrilecarboxylic acid ammonium salts were based on the initialconcentration of ω-nitrilecarboxylic acid ammonium salt present in thereaction mixture (unless otherwise noted), and determined by gaschromatography using a DB-1701 capillary column (30 m×0.53 mm ID, 1micron film thickness).

EXAMPLE 1 4-Cyanopentanoic Acid (Ammonium Salt)

First, 0.60 grams (wet cell weight) of frozen Acidovorax facilis 72 W(ATCC 55746) cells (previously heat-treated at 50° C. for 1 h beforefreezing) were placed into a 15-mL polypropylene centrifuge tube andfollowed by addition of 12 mL of potassium phosphate buffer (20 mM, pH7.0). After the cells were thawed and suspended, the resultingsuspension was centrifuged and the supernatant discarded. The resultingcell pellet was resuspended in a total volume of 12 mL of this samephosphate buffer. Into a second 15-mL polypropylene centrifuge tube wasweighed 0.1081 g (0.114 mL, 1.00 mmol, 0.100M) of2-methylglutaronitrile, then 9.89 mL of the A. facilis 72 W (ATCC 55746)cell suspension (0.494 g wet cell weight) was added and the resultingsuspension mixed on a rotating platform at 27° C. Samples (0.300 mL)were withdrawn and centrifuged, then 0.180 mL of the supernatant wasplaced in a Millipore Ultrafree-MC filter unit (10K MWCO) and mixed with0.020 mL of an aqueous solution of 0.750M N-methylpropionamide (HPLCexternal standard solution). Sufficient 1.0M HCl was added to lower thepH of the sample to ca. 2.5 and the resulting solution was filtered andanalyzed by HPLC. After 1.0 h, the HPLC yields of 4-cyanopentanoic acidand 2-methylglutaric acid were 99.3% and 0.7%, respectively, with no2-methylglutaronitrile remaining.

EXAMPLE 2 (COMPARATIVE) 4-Cyanopentanoic Acid (Ammonium Salt)

The procedure described in Example 1 was repeated using Acidovoraxfacilis 72 W (ATCC 55746) cells which had not been heat-treated at 50°C. for 1 h before freezing. After 1.0 h, the HPLC yields of4-cyanopentanoic acid and 2-methylglutaric acid were 62.7% and 34.6%.respectively with no 2-methyl-glutaronitrile remaining.

EXAMPLE 3 4-Cyanopentanoic Acid (Ammonium Salt)

First, 1.136 grams (wet cell weight) of frozen Acidovorax facilis 72 W(ATCC 55746) cells (previously heat-treated at 50° C. for 1 h beforefreezing) were placed into a 50-mL polypropylene centrifuge tube,followed by 21.6 mL of potassium phosphate buffer (20 mM, pH 7.0). Afterthe cells were thawed and suspended, the resulting suspension wascentrifuged and the supernatant discarded. The resulting cell pellet wasresuspended in a total volume of 22.7 mL of this same phosphate buffer.Into a 15-mL polypropylene centrifuge tube was weighed 0.4355 g (0.458mL, 4.00 mmol, 0.403M) of 2-methyl-glutaronitrile, then 9.54 mL of theA. facilis 72 W (ATCC 55746) cell suspension (0.477 g wet cell weight)was added and the resulting suspension mixed on a rotating platform at27° C. Samples (0.300 mL) were diluted 1:4 with distilled water, thencentrifuged, and 0.180 mL of the supernatant was placed in a MilliporeUltrafree-MC filter unit (10K MWCO) and mixed with 0.020 mL of anaqueous solution of 0.750M N-methylpropionamide (HPLC external standardsolution). Sufficient 1.0M HCl was added to lower the pH of the sampleto ca. 2.5 and the resulting solution was filtered and analyzed by HPLC.After 4.0 h, the HPLC yields of 4-cyanopentanoic acid and2-methylglutaric acid were 99.4% and 0.6%, respectively, with no2-methylglutaronitrile remaining.

EXAMPLE 4 4-Cyanopentanoic Acid (Ammonium Salt)

The procedure described in Example 3 was repeated, except that 1.086 g(1.143 mL, 10.04 mmol, two-phase reaction, 1.00M product) of2-methyl-glutaronitrile was mixed with 8.86 mL of the heat-treated A.facilis 72 W (ATCC 55746) cell suspension (0.443 g wet cell weight) wasmixed in a 15 mL polypropylene centrifuge tube on a rotating platform at27° C. Samples (0.300 mL) were diluted 1:10 with distilled water, thencentrifuged, and 0.180 mL of the supernatant was placed in a MilliporeUltrafree-MC filter unit (10 K MWCO) and mixed with 0.020 mL of anaqueous solution of 0.750M N-methylpropionamide (HPLC external standardsolution). Sufficient 1.0M HCl was added to lower the pH of the sampleto ca. 2.5, and the resulting solution was filtered and analyzed byHPLC. After 15.25 h, the HPLC yields of 4-cyanopentanoic acid and2-methylglutaric acid were 98.7% and 1.3%, respectively, with no2-methylglutaronitrile remaining.

EXAMPLE 5 4-Cyanopentanoic Acid (Ammonium Salt)

The procedure described in Example 1 was repeated using a suspension ofAcidovorax facilis mutant strain 72-PF-15 (ATCC 55747) which had notbeen heat-treated at 50° C. for 1 h. After 3.0 h, the HPLC yields of4-cyanopentanoic acid and 2-methylglutaric acid were 96.8% and 3.6%,respectively, with no 2-methylglutaronitrile remaining.

EXAMPLE 6 4-Cyanopentanoic Acid (Ammonium Salt)

The procedure described in Example 3 was repeated using a suspension ofAcidovorax facilis mutant strain 72-PF-15 (ATCC 55747) which had notbeen heat-treated at 50° C. for 1 h. A mixture of 0.4355 g (0.458 mL,4.00 mmol, 0.403M) of 2-methylglutaronitrile and 9.54 mL of the A.facilis mutant strain 72-PF-15 cell suspension (0.477 g wet cell weight)was mixed in a 15 mL polypropylene centrifuge tube on a rotatingplatform at 27° C. After 6.0 h, the HPLC yields of 4-cyanopentanoic acidand 2-methylglutaric acid were 98.8% and 1.2%, respectively, with no2-methylglutaronitrile remaining.

EXAMPLE 7 4-Cyanopentanoic Acid (Ammonium Salt)

The procedure described in Example 4 was repeated using a suspension ofAcidovorax facilis mutant strain 72-PF-15 (ATCC 55747) which had notbeen heat-treated at 50° C. for 1 h. A mixture of 1.086 g (1.143 mL,10.04 mmol, 1.00M of 2-methylglutaronitrile and 8.86 mL of the A.facilis mutant strain 72-PF-15 (ATCC 55747) cell suspension (0.443 g wetcell weight) was mixed in a 15 mL polypropylene centrifuge tube on arotating platform at 27° C. After 15.25 h, the HPLC yields of4-cyanopentanoic acid and 2-methylglutaric acid were 99.2% and 0.8%,respectively, with no 2-methylglutaronitrile remaining.

EXAMPLE 8 4-Cyanopentanoic Acid Isolation

Into a 2-L Erlenmeyer flask equipped with a magnetic stir bar wasweighed 150 g (wet cell weight) of frozen Acidovorax facilis 72 W (ATCC55746) (not previously heat-treated at 50° C. for 1 h before freezing).Potassium phosphate buffer (20 mM, pH 7.0) was then added to a totalvolume of 1.50 L. After the cells were thawed and suspended, theresulting suspension was heated in a water bath to 50° C. for 1 h, thencooled to 10° C. in an ice/water bath, centrifuged, and the supernatantdiscarded. The resulting cell pellet was washed once by resuspension in1.50 L of the same phosphate buffer, followed by centrifugation. Thewashed cell pellet was transferred to a 4-L erlenmeyer flask equippedwith magnetic stir bar, then suspended in a total volume of 2.5 L ofpotassium phosphate buffer (20 mM, pH 7.0). With stirring, 129.6 g(136.4 mL, 1.20 mol, 0.400M) of 2-methylglutaronitrile was added, andthe final volume adjusted to 3.00 L with the same phosphate buffer. Themixture was stirred at 25° C., and samples were withdrawn at regularintervals and analyzed by HPLC. After 21.5 h, the HPLC yields of4-cyanopentanoic acid (4-CPA) and 2-methylglutaric acid were 99.5% and0.5%, respectively, with no 2-methylglutaronitrile remaining.

The reaction mixture was centrifuged, the cell pellet recovered forreuse, and the resulting supernatant decanted and filtered using anAmicon 2.5 L Filter Unit equipped with a YM-10 filter (10K MWCO). Thefiltrate was placed in a 4.0 L flask, and the pH of the solutionadjusted to 2.5 with 6 N HCl. To the solution was then added sodiumchloride with stirring until saturated; then 1.0 L portions of theresulting solution were extracted with 4×500 mL of ethyl ether. Thecombined ether extracts were dried (MgSO₄), filtered, and the solutionconcentrated to 1.0 L by rotary evaporation at reduced pressure. To thesolution was then added 1.2 L of hexane and 0.200 mL of ethyl ether, andthe resulting solution cooled to -78° C. The resulting white crystallinesolid was isolated by rapid vacuum filtration and washing with 300 mL ofcold (5° C.) hexane. Residual solvent was removed under high vacuum (150millitorr) to yield 120.3 g (79% isolated yield) of 4-cyanopentanoicacid (mp. 31.9-32.6° C.).

EXAMPLE 9 4-Cyanopentanoic Acid (Ammonium Salt)

The recovered cell pellet from the Example 8 was reused in severalconsecutive 3.0-L batch reactions for the hydrolysis of2-methylglutaronitrile to 4-cyanopentanoic acid ammonium salt. Atconcentrations of 2-methylglutaronitrile greater than 0.400M, thesolubility of the dinitrile in the aqueous cell suspension was exceeded,and these reactions ran as two-phase aqueous/organic mixtures until theremaining 2-methylglutaronitrile was soluble in the reaction mixture.The Table below lists the final concentration of 4-CPA ammonium saltproduced, and the percent yields of 4-CPA and 2-methylglutaric acid.Based on dry weight of cell catalyst (1 gram wet weight=0.20 gram dryweight), 86 g 4-CPA/g dry weight of Acidovorax facilis 72 W (ATCC 55746)was produced.

    ______________________________________            Time    4-CPA(NH.sub.4)!                                4-CPA  2-MGA    rxn #   (h)    (VI)         (% yield)                                       (% yield)    ______________________________________    1       23     0.40         99.5   0.5    2       22     0.40         99.1   0.9    3       46     1.00         99.2   0.8    4       50     1.00         99.4   0.6    5       99     1.85         98.8   1.2    6       261    2.00         98.9   1.1    ______________________________________

EXAMPLE 10 3-Cyanopentanoic Acid (Ammonium Salt)

First, 0.60 grams (wet cell weight) of frozen Acidovorax facilis 72 W(ATCC 55746) (previously heat-treated at 50° C. for 1 h beforefreezing), was placed into a 15-mL polypropylene centrifuge tube, andthen followed by the addition of 10 mL of potassium phosphate buffer (20mM, pH 7.0). After the cells were thawed and suspended, the resultingsuspension was centrifuged, and the supernatant discarded. The resultingcell pellet was resuspended in a total volume of 10 mL of this samephosphate buffer. Into a second 15-mL polypropylene centrifuge tube wasweighed 0.1080 g (0.112 mL, 1.00 mmol, 0.100M of 2-ethylsuccinonitrile,8.00 mL of the A. facilis 72 W cell suspension (0.5 g wet cell weight)was added, the total volume adjusted to 10.0 mL with potassium phosphatebuffer (20 mM, pH 7.0), and the resulting suspension mixed on a rotatingplatform at 27° C. Samples (0.300 mL) were withdrawn and centrifuged,then 0.180 mL of the supernatant was placed in a Millipore Ultrafree-MCfilter unit (0.22 micron) and mixed with 0.020 mL of an aqueous solutionof 0.750M N-methylpropionamide (HPLC external standard solution). Theresulting solution was filtered and analyzed by HPLC. After 1.0 h, theHPLC yield of 3-cyanopentanoic acid was 100%, with no2-ethylsuccinonitrile remaining, and no other byproducts observed.

EXAMPLE 11 3-Cyanopentanoic Acid (Ammonium Salt)

The procedure described in Example 10 was repeated, using 0.4337 g(0.449 mL, 4.01 mmol, 0.401M) of 2-ethylsuccinonitrile and 8.00 mL ofthe heat-treated A. facilis 72 W (ATCC 55746) cell suspension (0.5 g wetcell weight), in a total volume of 10.0 mL (adjusted with potassiumphosphate buffer (20 mM, pH 7.0)). Samples (0.100 mL) were withdrawn,diluted 1:4 with water and centrifuged; 0.180 mL of the supernatant wasthen placed in a Millipore Ultrafree-MC filter unit (0.22 micron) andmixed with 0.020 mL of an aqueous solution of 0.750MN-methylpropionamide (HPLC external standard solution). The resultingsolution was tiltered and analyzed by HPLC. After 8.0 h. the HPLC yieldot 3-cyanopentanoic acid was 100%, with no 2-ethylsuccinonitrileremaining and no other byproducts observed.

EXAMPLE 12 3-Cyanopentanoic Acid (Ammonium Salt)

The procedure described in Example 10 was repeated, using 1.087 g (1.13mL, 10.1 mmol, two-phase reaction, 1.01M product) of2-ethylsuccinonitrile and 8.00 mL of the heat-treated A. facilis 72 W(ATCC 55746) cell suspension (0.5 g wet cell weight), in a total volumeof 10.0 mL (adjusted with potassium phosphate buffer (20 mM, pH 7.0)).Samples (0.100 mL) were withdrawn, diluted 1:10 with water andcentrifuged, then 0.180 mL of the supernatant was placed in a MilliporeUltrafree-MC filter unit (0.22 micron) and mixed with 0.020 mL of anaqueous solution of 0.750M N-methylpropionamide (HPLC external standardsolution). The resulting solution was filtered and analyzed by HPLC.After 71 h, the HPLC yield of 3-cyanopentanoic acid was 100%, with no2-ethylsuccinonitrile remaining, and no other byproducts observed.

EXAMPLE 13 3-Cyanopentanoic Acid Isolation

Into a 4.0 L erlenmeyer flask equipped with magnetic stir bar was placed161 g of frozen Acidovorax facilis 72 W (ATCC 55746) cells (previouslyheat-treated at 50° C. for 1 h before freezing) and 1.60 L of potassiumphosphate buffer (20 mM, pH 7.0) at 27° C. With stirring, the cells werethawed and suspended, then 325 g (336.0 mL, 3.00 mole) of2-ethylsuccinonitrile was added with stirring, and the total volume ofthe mixture adjusted to 2.40 L with potassium phosphate buffer (20 mM,pH 7.0). The resulting mixture was stirred at 27° C., and samples (0.100mL) were withdrawn, diluted 1:10 with water and centrifuged, 0.180 mL ofthe supernatant was then placed in a Millipore Ultrafree-MC filter unit(0.22 micron) and mixed with 0.020 mL of an aqueous solution of 0.750MN-methylpropionamide (HPLC external standard solution). The resultingsolution was filtered and analyzed by HPLC. After 183 h, the HPLC yieldof 3-cyanopentanoic acid was 100%, with no 2-ethylsuccinonitrileremaining, and no other byproducts observed. The reaction mixture wascentrifuged, the cell pellet recovered for reuse, and the resulting 2.3L of supernatant decanted and filtered using an Amicon 2.5 L Filter Unitequipped with a YM-10 filter (10K MWCO). The concentration of3-cyanopentanoic acid ammonium salt in the filtrate was 1.26M.

A 300-mL portion of the filtrate described above, containing 1.26M3-cyanopentanoic acid ammonium salt, was adjusted to pH 2.5 with ca. 60mL of 6N HCl, then saturated with sodium chloride and extracted with4×200 mL of ethyl ether. The combined organic extracts were dried overmagnesium sulfate, filtered, and the solvent removed by rotaryevaporation at reduced pressure at 28° C. The resulting slightly-yellowviscous oil was stirred under high vacuum (50 millitorr) to removeresidual solvent at 28° C., then cooled to -20° C. for 2-3 h to produce3-cyanopentanoic acid as a crystalline white solid (45.9 g, 96% yield);m.p. 33.0-34.0° C.

EXAMPLE 14 3-Cyanopentanoic Acid (Ammonium Salt)

The procedure described in Example 10 was repeated using a suspension ofAcidovorax facilis mutant strain 72-PF-15 (ATCC 55747) which had notbeen heat-treated at 50° C. After 1.0 h, the HPLC yield of3-cyanopentanoic acid was 100%, with no 2-ethylsuccinonitrile remaining,and no other byproducts observed.

EXAMPLE 15 3-Cyanopentanoic Acid (Ammonium Salt)

The procedure described in Example 14 was repeated using 0.4325 g (0.455mL, 4.00 mmol, 0.400M of 2-ethylsuccinonitrile and 8.00 mL (0.5 g wetcell weight) of a suspension of Acidovorax facilis mutant strain72-PF-15 (ATCC 55747) which had not been heat-treated at 50° C., in atotal volume of 10.0 mL (adjusted with potassium phosphate buffer (20mM, pH 7.0)). After 25 h, the HPLC yield of 3-cyanopentanoic acid was100%, with no 2-ethylsuccinonitrile remaining, and no other byproductsobserved.

EXAMPLE 16 3-Cyanopentanoic Acid (Ammonium Salt)

The procedure described in Example 14 was repeated, using 1.087 g (1.14mL, 10.1 mmol, two-phase reaction, 1.01M product) of2-ethylsuccinonitrile and 8.00 mL (0.5 g wet cell weight) of asuspension of Acidovorax facilis mutant strain 72-PF-15 (ATCC 55747)which had not been heat-treated at 50° C., in a total volume of 10.0 mL(adjusted with potassium phosphate buffer (20 mM, pH 7.0)). After 71 h,the HPLC yield of 3-cyanopentanoic acid was 76.6%, with 25.1%2-ethylsuccinonitrile remaining, and no other byproducts observed.

EXAMPLE 17 4-Cyano-4-Pentenoic Acid Isolation

Into a 500 mL erlenmeyer flask equipped with magnetic stir bar wasplaced 50.0 g of frozen Acidovorax facilis 72 W (ATCC 55746) cells(previously heat-treated at 50° C. for 1 h before freezing) and 450 mLof potassium phosphate buffer (20 mM, pH 7.0) at 27° C. With stirring,the cells were thawed and suspended, then centrifuged and thesupernatant discarded. The cell pellet was resuspended in a total volumeof 863 mL ot potassium phosphate buffer (20 mM, pH 7.0) in a 1.0 Lflask. then 133 g (137.0 mL. 1.25 mole) of 2-methyleneglutaronitrile wasadded with stirring at 27° C. At concentrations of2-methyleneglutaronitrile greater than 0.400M, the solubility of thedinitrile in the aqueous cell suspension was exceeded, and thesereactions ran as two-phase aqueous/organic mixtures until the remaining2-methyleneglutaronitrile was soluble in the reaction mixture. Samples(0.100 mL) were withdrawn, diluted 1:10 with water and centrifuged, then0.150 mL of the supernatant was placed in a Millipore Ultrafree-MCfilter unit (0.22 micron) and mixed with 0.150 mL of an aqueous solutionof 0.150M N-methylpropionamide (HPLC external standard solution). Theresulting solution was filtered and analyzed by HPLC. After 26 h, theHPLC yield of 4-cyano-4-pentenoic acid was 100%, with no2-methyleneglutaronitrile remaining, and no other byproducts observed.The reaction mixture was centrifuged, the cell pellet was recovered forreuse, and the supernatant was filtered using an Amicon 2.5 L FilterUnit equipped with a YM-10 filter (10K MWCO). The final concentration of4-cyano-4-pentenoic acid ammonium salt in the filtrate was 1.298M.

A 100-mL portion of the filtrate containing the 4-cyano-4-pentenoic acidammonium salt product mixture described above was adjusted to pH 2.7with 6N HCl, then saturated with sodium chloride and extracted with4×100 mL of ethyl ether. The combined organic extracts were dried overmagnesium sulfate, filtered, and the volume of the combined extractsreduced to 100 mL by rotary evaporation at reduced pressure at 28° C. Tothe ether solution was added 200 mL of hexane, and the resultingsolution cooled to -78° C. The resulting white solid which crystallizedwas isolated by rapid vacuum filtration and washing with 100 mL of cold(5° C.) hexane. Residual solvent was removed under high vacuum (150millitorr) to yield 9.80 g (60% isolated yield) of 4-cyano-4-pentenoicacid (m.p. 26.5-27.0° C., stored at -20° C.).

EXAMPLE 18 4-Cyano-4-Pentenoic (Ammonium Salt)

The recovered cell pellet from the Example 17 (Acidovorax facilis 72 W(ATCC 55746) cells) was reused in a second consecutive 1.0-L batchreactions for the hydrolysis of 2-methyleneglutaronitrile to4-cyano-4-pentenoic acid ammonium salt. At concentrations of2-methyleneglutaronitrile greater than 0.400M, the solubility of thedinitrile in the aqueous cell suspension was exceeded, and thesereactions ran as two-phase aqueous/organic mixtures until the remaining2-methyleneglutaronitrile was soluble in the reaction mixture. The cellpellet was resuspended in a total volume of 781 ml of potassiumphosphate buffer (20 mM, pH 7.0) in a 1.0 L flask, then 212 g (219.0 mL,2.00 mole) of 2-methyleneglutaronitrile was added with stirring at 27°C. Samples (0.100 mL) were withdrawn, diluted 1:20 with water andcentrifuged then 0.150 mL of the supernatant was placed in a MilliporeUltrafree-NIC filter unit (0.22 micron) and mixed with 0.150 mL of anaqueous solution of 0.150M N-methylpropionamide (HPLC external standardsolution). The resulting solution was filtered and analyzed by HPLC.After 53 h, the HPLC yield of 4-cyano-4-pentenoic acid was 100%, with no2-methyleneglutaronitrile remaining, and no other byproducts observed.

EXAMPLE 19 3-Cyanopropanoic Acid (Ammonium Salt)

First, 0.50 grams (wet cell weight) of frozen Acidovorax facilis 72 W(ATCC 55746) (previously heat-treated at 50° C. for 1 h before freezing)were placed into a 15-mL polypropylene centrifuge tube and followed bythe addition of 10 mL of potassium phosphate buffer (20 mM, pH 7.0).After the cells were thawed and suspended, the resulting suspension wascentrifuged, and the supernatant discarded. The resulting cell pelletwas resuspended in a total volume of 10 mL of this same phosphatebuffer. Into a second 15-mL polypropylene centrifuge tube was placed0.3236 g (4.00 mmol, 0.400M of succinonitrile dissolved in a totalvolume of 8.0 mL of potassium phosphate buffer (20 mM, pH 7.0), then2.00 mL of the A. facilis 72 W (ATCC 55746) cell suspension (0.10 g wetcell weight) was added, and the resulting suspension mixed on a rotatingplatform at 27° C. Samples (0.100 mL) were withdrawn, diluted 1:4 withwater and centrifuged, then 0.150 mL of the supernatant was placed in aMillipore Ultrafree-MC filter unit (0.22 micron) and mixed with 0.015 mLof 0.1M HCl and 0.150 mL of an aqueous solution of 0.100M propionic acid(HPLC external standard solution). The resulting solution was filteredand analyzed by HPLC. After 3.0 h, the HPLC yield of 3-cyanopropanoicacid was 100%, with no succinonitrile remaining, and no other byproductsobserved.

EXAMPLE 20 3-Cyanopropanoic Acid Isolation

Into a 500 mL erlenmeyer flask equipped with magnetic stir bar wasplaced 20.0 g of frozen Acidovorax facilis 72 W (ATCC 55746) cells(heat-treated at 50° C. for 1 h before freezing) and 200 mL of potassiumphosphate buffer (20 mM, pH 7.0) at 27° C. With stirring, the cells werethawed and suspended, then centrifuged and the supernatant discarded.This wash procedure was repeated. The resulting cell pellet wasresuspended in a total volume of 1.00 L of potassium phosphate buffer(20 mM, pH 7.0) containing 101.1 g (1.25 mole, 1.25M) succinonitrile andthe resulting mixture was stirred in a 1-L flask placed in a water bathat 27° C. Samples (0.100 mL) were withdrawn, diluted 1:10 with water andcentrifuged. then 0.150 mL of the supernatant was placed in a MilliporeUltratree-MC filter unit (0.22 micron) and mixed with 0.0 15 mL ot 0.1MHCl and 0.150 mL of an aqueous solution of 0.100M propionic acid (HPLCexternal standard solution). The resulting solution was filtered andanalyzed by HPLC. After 1.0 h, the HPLC yield of 3-cyanopropanoic acidand succinic acid were 99.7% and 0.3%, respectively, with nosuccinonitrile remaining. The reaction mixture was centrifuged, and thesupernatant was filtered using an Amicon 2.5 L Filter Unit equipped witha YM-10 filter (10K MWCO). The final concentration of 3-cyanopropanoicacid ammonium salt in the filtrate was 1.31M.

A 200-mL portion of the filtrate containing the 3-cyanopropanoic acidammonium salt product mixture described above was adjusted to pH 2.5with 6N HCl, then saturated with sodium chloride and extracted with4×200 mL of ethyl ether. The combined organic extracts were dried overmagnesium sulfate, filtered, and the solvent removed by rotaryevaporation at reduced pressure. The resulting colorless oil wasdissolved in 150 mL of ethyl ether, then 100 mL of hexane was added, andthe resulting solution cooled to -78° C. The resulting white solid whichcrystallized was isolated by vacuum filtration, and residual solvent wasremoved under high vacuum (150 millitorr) to yield 14.32 g (55% isolatedyield) of 3-cyanopropanoic acid (m.p. 49.5-51.0° C.).

EXAMPLE 21 4-Cyanobutyric Acid (Ammonium Salt)

First, 0.50 grams (wet cell weight) of frozen Acidovorax facilis 72 W(ATCC 55746) (previously heat-treated at 50° C. for 1 h before freezing)were placed into a 15-mL polypropylene centrifuge tube and then followedby the addition of 10 mL of potassium phosphate buffer (20 mM, pH 7.0).After the cells were thawed and suspended, the resulting suspension wascentrifuged, and the supernatant discarded. The resulting cell pelletwas resuspended in a total volume of 10 mL of this same phosphatebuffer. Into a second 15-mL polypropylene centrifuge tube was placed0.3830 g (4.03 mmol, 0.400M of glutaronitrile dissolved in a totalvolume of 6.0 mL of potassium phosphate buffer (20 mM, pH 7.0), then4.00 mL of the A. facilis 72 W (ATCC 55746) cell suspension (0.20 g wetcell weight) was added, and the resulting suspension mixed on a rotatingplatform at 27° C. Samples (0.100 mL) were withdrawn, diluted 1:4 withwater and centrifuged, then 0.150 mL of the supernatant was placed in aMillipore Ultrafree-MC filter unit (0.22 micron) and mixed with 0.015 mLof 0.1M HCl and 0.150 mL of an aqueous solution of 0.150M potassiumacetate (HPLC external standard solution). The resulting solution wasfiltered and analyzed by HPLC. After 4.0 h, the HPLC yield of4-cyanobutyric acid and glutaric acid were 85.1% and 8.2%, respectively,with 5.7% glutaronitrile remaining.

EXAMPLE 22 4-Cyanobutyric Acid Isolation

Into a 250 mL erlenmeyer flask equipped with magnetic stir bar wasplaced 15.0 g of frozen Acidovorax facilis 72 W (ATCC 55746) cells(heat-treated at 50° C. for 1 h before freezing) and 135 mL of potassiumphosphate buffer (20 mM, pH 7.0) at 27° C. With stirring, the cells werethawed and suspended, then centrifuged and the supernatant discarded.This wash procedure was repeated. The resulting cell pellet wasresuspended in a total volume of 100 mL of potassium phosphate buffer(20 mM, pH 7.0) and this cell suspension was added to a 500 mL flaskcontaining a magentic stir bar, 42.78 g (0.450 mole) of glutaronitrileand 157.2 mL of potassium phosphate buffer (20 mM, pH 7.0). Theresulting mixture, containing 1.5M glutaronitrile, was stirred at 27° C.Samples (0.100 mL) were withdrawn, diluted 1:10 with water andcentrifuged. Then, 0.200 mL of the supernatant was placed in a MilliporeUltrafree-MC filter unit (0.22 micron) and mixed with 0.020 mL of 0.1MHCl and 0.200 mL of an aqueous solution of 0.150M potassium acetate(HPLC external standard solution). The resulting solution was filteredand analyzed by HPLC. After 4.0 h, the HPLC yield of 4-cyanobutyric acidammonium salt and glutaric acid diammonium salt were 92.3% and 7.7%,respectively, with no glutaronitrile remaining.

The reaction mixture was centrifuged and the supernatant was filteredusing an Amicon 2.5 L Filter Unit equipped with a YM-10 filter (10KMWCO). The filtrate containing the 4-cyanobutyric acid ammonium saltproduct mixture described above was adjusted to pH 3.5 with 6N HCl, thensaturated with sodium chloride and extracted with 4×300 mL of ethylether. The combined organic extracts were dried over magnesium sulfate,filtered, and the solvent removed by rotary evaporation at reducedpressure followed by stirring under vacuum (100 millitorr) to yield 35.3g (62% yield) of 4-cyanobutyric acid as a pale yellow oil whichcrystallized upon standing. The solid was recrystallized from 1:1 ethylacetate/hexane at 5° C. (mp. 39.6-40.2° C.).

EXAMPLE 23 5-Cyanopentanoic Acid (Ammonium Salt)

First, 2.0 grams (wet cell weight) of frozen Comamonas testosteroni5-MGAM-4 D (ATCC 55744) cells (previously heat-treated at 50° C. for 1 hbefore freezing) were placed into a 50-mL polypropylene centrifuge tubeand followed by the addition of 30 mL of potassium phosphate buffer (20mM, pH 7.0). After the cells were thawed and suspended, the resultingsuspension was centrifuged, and the supernatant discarded. The resultingcell pellet was resuspended in a total volume of 30 mL of this samephosphate buffer. Into a second 15-mL polypropylene centrifuge tube wasweighed 0.1085 g (0.114 mL, 1.00 mmol, (0.100M) of adiponitrile, then9.29 mL of potassium phosphate buffer (20 mM, pH 7.0) and 0.60 mL of theComamonas testosteroni 5-MGAM-4 D (ATCC 55744) cell suspension (0.040 gwet cell weight) was added, and the resulting suspension mixed on arotating platform at 27° C. Samples (0.300 mL) were withdrawn andcentrifuged. then 0.180 mL of the supernatant was placed in a MilliporeUltrafree-MC filter unit (10K MWCO) and mixed with 0.020 mL of anaqueous solution of 0.750M N-ethylacetamide (HPLC external standardsolution) and sufficient 1.0M HCL to lower the pH of the sample to ca.pH 2.5. The resulting solution was filtered and analyzed by HPLC. After5.0 h, the HPLC yield of 5-cyanopentanoic acid, adipamic acid,adipamide, and adipic acid were 96.6%, 1.7%, 1.4% and 0.3%, with noadiponitrile remaining.

EXAMPLE 24 5-Cyanopentanoic Acid (Ammonium Salt)

The procedure described in Example 23 was repeated, using 0.4338 g(0.457 mL, 4.01 mmol, 0.401M) of adiponitrile and 3.0 mL of theheat-treated Comamonas testosteroni 5-MGAM-4 D (ATCC 55744) cellsuspension (0.20 g wet cell weight), in a total volume of 10.0 mL(adjusted with potassium phosphate buffer (20 mM, pH 7.0)). Samples(0.100 mL) were withdrawn, diluted 1:4 with water and centrifuged, then0.180 mL of the supernatant was placed in a Millipore Ultrafree-MCfilter unit (10K MWCO) and mixed with 0.020 mL of an aqueous solution of0.750M N-ethylacetamide (HPLC external standard solution) and sufficient1.0M HCl to lower the pH of the sample to ca. pH 2.5. The resultingsolution was filtered and analyzed by HPLC. After 5.0 h, the HPLC yieldof 5-cyanopentanoic acid, adipamic acid, adipamide, and adipic acid were94.0%, 4.0%, 0.6% and 1.4%, with no adiponitrile remaining.

EXAMPLE 25 5-Cyanopentanoic Acid (Ammonium Salt)

The procedure described in Example 23 was repeated, using 1.084 g (1.14mL, 10.0 mmol, two-phase reaction, 1.00M product) of adiponitrile and7.5 mL of the heat-treated Comamonas testosteroni 5-MGAM-4 D (ATCC55744) cell suspension (0.50 g wet cell weight), in a total volume of10.0 mL (adjusted with potassium phosphate buffer (20 mM, pH 7.0)).Samples (0.100 mL) were withdrawn, diluted 1:10 with water andcentrifuged, then 0.180 mL of the supernatant was placed in a MilliporeUltrafree-MC filter unit (10K MWCO) and mixed with 0.020 mL of anaqueous solution of 0.750M N-ethylacetamide (HPLC external standardsolution) and sufficient 1.0M HCl to lower the pH of the sample to ca.pH 2.5. The resulting solution was filtered and analyzed by HPLC. After24.0 h, the HPLC yield of 5-cyanopentanoic acid, adipamic acid,adipamide. and adipic acid were 90.0%, 4.2%, 0.0% and 5.7%, with noadiponitrile remaining.

EXAMPLE 26 5-Cyanopentanoic Acid Isolation

Into a 2-L erlenmeyer flask equipped with magnetic stir bar was placed asuspension of 4.0 g of Comamonas testosteroni 5-MGAM-4 D (ATCC 55744)cells (previously heat-treated at 50° C. for 1 h) in 1.72 L of potassiumphosphate buffer (20 mM, pH 7.0) at 27° C. To the suspension was thenadded with stirring 270.4 g (284.3 mL, 2.5 mole, ca. 1.25M final productconcentration) of adiponitrile, and the resulting mixture was stirred at27° C. Samples (0.200 mL) were withdrawn, diluted 1:5 with water andcentrifuged, then 0.200 mL of the supernatant was placed in a MilliporeUltrafree-MC filter unit (10K MWCO) and sufficient 1.0M HCl added tolower the pH of the sample to ca. pH 2.5. The resulting solution wasfiltered and analyzed by HPLC. After 63 h, the hydrolysis reaction hadslowed considerably, so an additional 10.0 g of the same microbial cellcatalyst was added to the mixture. After 86 h, the HPLC yield of5-cyanopentanoic acid, adipamic acid, adipamide, and adipic acid were88.2%, 4.7%, 6.6% and 0.0%, with no adiponitrile remaining. The reactionmixture was centrifuged, and the supernatant was filtered using anAmicon 2.5 L Filter Unit equipped with a YM-10 filter (10K MWCO).

A 200-mL portion of the filtrate of the product mixture described above,containing the 5-cyanopentanoic acid ammonium salt (1.13M), was adjustedto pH 2.5 with 6N HCl, then saturated with sodium chloride and extractedwith 4×200 mL of ethyl ether. The combined ether extracts were driedover magnesium sulfate, filtered, and the solvent removed by rotaryevaporation at reduced pressure. Remaining ether was removed by stirringthe colorless liquid at room temperature under high vacuum (60millitorr) for 5 h to yield 27.32 g (95% isolated yield) of5-cyanopentanoic acid. The 5-cyanopentanoic acid was then distilledunder vacuum (75 millitorr) at 110-112° C. without decomposition.

EXAMPLE 27 5-Methyl-2-Piperidone from 4-Cyanopentanoic Acid AmmoniumSalt

Into a 100 mL graduated cylinder was placed 54.4 mL of an aqueousreaction mixture containing 1.85M 4-cyanopentanoic acid ammonium salt(0.1 mole 4-cyanopentanoic acid ammonium salt, produced by the enzymatichydrolysis of 2-methylglutaronitrile; Example 9, filtered productmixture from reaction #5), then 12.9 mL of concentrated ammoniumhydroxide (29.3% NH₃, 0.2 mole NH₃) was added and the final volumeadjusted to 100 mL with distilled water. The final concentrations of4-cyanopentanoic acid ammonium salt and added ammonium hydroxide were1.0M and 2.0M, respectively. To the resulting solution was added 0.631 g(5 wt. %/wt. of 4-cyanopentanoic acid) at chromium-promoted Raney Nickel(Grace Davison Raney® 2400 Active Metal Catalyst), and the resultingmixture charged to a 300-mL 314 SS Autoclave Engineers EZE-Seal stirredautoclave equipped with a Dispersimax® turbine-type impeller. Afterflushing the reactor with nitrogen, the contents of the reactor werestirred at 1000 rpm and heated at 160° C. under 500 psig of hydrogen gasfor 3 h. After cooling to room temperature, analysis of the finalreaction mixture by gas chromatography indicated a 96.4% yield of5-methyl-2-piperidone, with no 4-cyanopentanoic acid ammonium saltremaining.

The product mixture was filtered to remove the catalyst, then adjustedto pH 6.0 with 6N HCl and saturated with sodium chloride. The resultingsolution was extracted five times with 100 mL of dichloro-methane, andthe combined organic extracts dried over magnesium sulfate, filtered,and the solvent removed by rotary evaporation under reduced pressure toyield a colorless oil. After removal of the remaining solvent undervacuum (0.1 mm Hg), the oil crystallized to form a white solid, whichwas recrystallized from 150 mL of ethyl ether at -78° C. to yield 6.69 g(59% isolated yield) of 5-methyl-2-piperidone (mp 55.5-56.2° C.).

EXAMPLE 28 5-Methyl-2-Piperidone from 4-Cyanopentanoic Acid

The reaction described in Example 27 was repeated using 12.71 g (0.100mole) of crystalline 4-cyanopentanoic acid (from an isolation describedin Example 8) and 19.34 mL of concentrated ammonium hydroxide (29.3%NH₃, 0.3 mole NH₃) in a total volume of 100 mL. The final concentrationsof 4-cyanopentanoic acid ammonium salt and ammonia were 1.0M and 2.0M,respectively. Analysis of the final reaction mixture by gaschromatography indicated a 91.1% yield of 5-methyl-2-piperidone, with no4-cyanopentanoic acid remaining.

EXAMPLE 29 5-Methyl-2-Piperidone from 4-Cyanopentanoic Acid AmmoniumSalt

Hydrogenations of 5-mL aqueous reaction mixtures containing 1.0M4-cyanopentanoic acid ammonium salt (filtered product mixture fromExample 9, reaction #5), 0 to 3.0M ammonium hydroxide, and 5 wt. % or 10wt. % (relative to weight of 4-cyanopentanoic acid) of a hydrogenationcatalyst selected from a group consisting of Cr-promoted Raney nickelcatalyst (Raney 2400), 5% palladium on carbon, 10% palladium on carbon,5% ruthenium on alumina, or 10% ruthenium on alumina were run in glassshaker tubes at 500 psig hydrogen gas and at either 160° C. or 180° C.were examined for the production of the corresponding lactam5-methyl-2-piperidone (5-MPPD):

    ______________________________________                                         %                                         4-CPA 5-    Temp.             NH.sub.4 OH!                              wt. % Time con-  MPPD    (° C.)          catalyst   (M)      catalyst                                    (h)  version                                               (%)    ______________________________________    160   Raney 2400 0        5     2    100   85.6    160   Raney 2400 2.0      5     3    100   96.4    160   Raney 2400 3.0      5     2    100   86.5    160   5% Pd/C    2.0      5     2    10    0    160   10% Pd/C   2.0      5     2    14    0    160   5% Ru/Al2O3                     2.0      5     2    15    0    160   10% Ru/Al2O3                     2.0      5     2    20    0    180   Raney 2400 2.0      5     2    100   91.4    180   Raney 2400 3.0      5     2    100   89.5    ______________________________________

EXAMPLE 30 4-Ethylpyrrolidin-2-one from 3-Cyanopentanoic Acid AmmoniumSalt

Into a 100 mL graduated cylinder was placed 79.4 mL of an aqueousreaction mixture containing 1.26M 3-cyanopentanoic acid ammonium salt(0.1 mole 3-cyanopentanoic acid ammonium salt, produced by the enzymatichydrolysis of 2-ethylsuccinonitrile; Example 13 filtered productmixture), then 12.9 mL of concentrated ammonium hydroxide (29.3% NH₃,0.2 mole NH₃) was added and the final volume adjusted to 100 mL withdistilled water. The final concentrations of 3-cyanopentanoic acidammonium salt and added ammonium hydroxide were 1.0M and 2.0M,respectively. To the resulting solution was added 0.631 g (5 wt. %/wt.of 3-cyano-pentanoic acid) of chromium-promoted Raney Nickel (GraceDavison Raney® 2400 Active Metal Catalyst), and the resulting mixturecharged to a 300-mL 314 SS Autoclave Engineers EZE-Seal stirredautoclave equipped with a Dispersimax® turbine-type impeller. Afterflushing the reactor with nitrogen, the contents of the reactor werestirred at 1000 rpm and heated at 160° C. under 500 psig of hydrogen gasfor 4 h. After cooling to room temperature, analysis of the finalreaction mixture by gas chromatography indicated a 90.7% yield of4-ethylpyrrolidin-2-one, with no 3-cyanopentanoic acid ammonium saltremaining.

EXAMPLE 31 4-Ethylpyrrolidin-2-one from 3-Cyanopentanoic Acid

The reaction described in Example 30 was repeated using 12.71 g (0.100mole) of crystalline 3-cyanopentanoic acid (from isolation described inExample 13) and 19.34 mL of concentrated ammonium hydroxide (29.3% NH₃,0.3mole NH₃) in a total volume of 100 mL. The final concentrations of3-cyanopentanoic acid ammonium salt and added ammonium hydroxide were1.0M and 2.0M, respectively. Analysis of the final reaction mixture (2 hreaction time) by gas chromatography indicated a 92.1% yield of4-ethylpyrrolidin-2-one. with no 3-cyanopentanoic acid remaining.

The product mixture was filtered to remove the catalyst, then adjustedto pH 7.0 with 6N HCl and saturated with sodium chloride. The resultingsolution was extracted four times with 100 mL of dichloro-methane, andthe combined organic extracts dried over magnesium sulfate, filtered,and the solvent removed by rotary evaporation under reduced pressure toyield a colorless oil. After removal of the remaining solvent undervacuum (0.1 mm Hg), the oil was dissolved in 150 mL of ethyl ether,which was then cooled to -78° C. After 1 h, the white solid which hadcrystallized was collected by vacuum filtration to yield a total of 8.96g (79% isolated yield) of 4-ethylpyrrolidin-2-one (mp 40.5-41.5° C.).

EXAMPLE 32 4-Ethylpyrrolidin-2-one from 3-Cyanopentanoic Acid AmmoniumSalt

The reaction described in Example 30 was repeated exactly as describedexcept that the temperature employed for the hydrogenation was 140° C.Analysis of the final reaction mixture (4 h reaction time) by gaschromatography indicated a 87.2% yield of 4-ethylpyrrolidin-2-one, withno 3-cyanopentanoic acid remaining.

EXAMPLE 33 4-Ethylpyrrolidin-2-one from 3-Cyanopentanoic Acid AmmoniumSalt

The reaction described in Example 30 was repeated exactly as describedexcept that 1.262 g (10 wt. %/wt. of 3-cyano-pentanoic acid) ofchromium-promoted Raney Nickel (Grace Davison Raney® 2400 Active MetalCatalyst) was employed. Analysis of the final reaction mixture (1.5 hreaction time) by gas chromatography indicated a 91.0% yield of4-ethylpyrrolidin-2-one, with no 3-cyanopentanoic acid remaining.

EXAMPLE 34 4-Ethylpyrrolidin-2-one from 3-Cyanopentanoic Acid AmmoniumSalt (Temperature Dependence)

Hydrogenations of 5-mL aqueous reaction mixtures containing 1.0M3-cyanopentanoic acid (3-CPA) ammonium salt (filtered product mixturefrom Example 13), 2.0M ammonium hydroxide, and 5 wt. % or 10 wt. % ofCr-promoted Raney nickel catalyst (Raney 2400) (relative to weight of3-cyanopentanoic acid) were run in glass shaker tubes at 500 psighydrogen gas and at temperatures from 70° C. to 180° C. for 2 h, thenanalyzed by high pressure liquid chromatography for conversion of 3-CPAand by gas chromatography for the production of 4-ethylpyrrolidin-2-one:

    ______________________________________          3-CPA          ammo-          nium              wt. %    Temp. salt      NH.sub.4 OH!                            Raney Time 3-CPA  4-EPRD    (° C.)          (M)      (M)      Ni    (h)  (% conv.)                                              (% yield)    ______________________________________    70    1.0      2.0      5     2    7      1.5    120   1.0      2.0      5     2    55     22.7    140   1.0      2.0      5     2    71     55.4    140   1.0      2.0      10    2    100    89.9    160   1.0      2.0      5     2    100    90.1    160   1.0      2.0      10    2    100    91.3    180   1.0      2.0      5     2    100    86.1    180   1.0      2.0      10    2    100    90.0    ______________________________________

EXAMPLE 35 4-Ethylpyrrolidin-2-one from 3-Cyanopentanoic Acid AmmoniumSalt (NH₄ OH Concentration Dependence)

Hydrogenations of 5-mL aqueous reaction mixtures containing 1.0M3-cyanopentanoic acid (3-CPA) ammonium salt (filtered product mixturefrom Example 13), from 0M to 3.0M ammonium hydroxide, and 5 wt. % ofCr-promoted Raney nickel catalyst (Raney 2400) (relative to weight of3-cyanopentanoic acid) were run in glass shaker tubes at 500 psighydrogen gas and at temperatures of 160° C. or 180° C. for 2 h, thenanalyzed by high pressure liquid chromatography for conversion of 3-CPAand by gas chromatography for the production of 4-ethylpyrrolidin-2-one:

    ______________________________________          3-CPA          ammo-          nium              wt. %    Temp. salt      NH.sub.4 OH!                            Raney Time 3-CPA  4-EPRD    (° C.)          (M)      (M)      Ni    (h)  (% conv.)                                              (% yield)    ______________________________________    160   1.0      0        5     2    99     80.1    160   1.0      1.0      5     2    99     87.6    160   1.0      2.0      5     2    100    90.1    160   1.0      3.0      5     2    100    85.4    180   1.0      0        5     2    100    75.1    180   1.0      1.0      5     2    100    85.8    180   1.0      2.0      5     2    100    88.5    180   1.0      3.0      5     2    100    90.0    ______________________________________

EXAMPLE 36 4-Ethylpyrrolidin-2-one from 3-Cyanopentanoic Acid AmmoniumSalt (3-CPA Concentration Dependence)

Hydrogenations of 5-mL aqueous reaction mixtures containing 1.0M, 1.5M,or 2.0M 3-cyanopentanoic acid (3-CPA) (isolated from filtered productmixture, Example 13), and 2.0M, 3.0M, or 4.0M ammonium hydroxide,respectively, were run using as catalyst 5 wt. % of Cr-promoted Raneynickel (Raney 2400) (relative to weight of 3-cyanopentanoic acid) inglass shaker tubes at 500 psig hydrogen gas and at temperatures of 160°C. or 180° C. for 2 h, then analyzed by high pressure liquidchromatography for conversion of 3-CPA and by gas chromatography for theproduction of 4-ethylpyrrolidin-2-one:

    ______________________________________          3-CPA          ammo-          nium              wt. %    Temp. salt      NH.sub.4 OH!                            Raney Time 3-CPA  4-EPRD    (° C.)          (M)      (M)      Ni    (h)  (% conv.)                                              (% yield)    ______________________________________    160   1.0      2.0      5     2    100    90.1    160   1.5      3.0      5     2    99     76.1    160   2.0      4.0      5     2    99     80.0    180   1.0      2.0      5     2    100    88.5    180   1.5      3.0      5     2    99     77.3    180   2.0      4.0      5     2    99     84.1    ______________________________________

EXAMPLE 37 Caprolactam from 5-Cyanopentanoic Acid Ammonium Salt

Hydrogenations of 5-mL aqueous reaction mixtures containing 1.0M5-cyanopentanoic acid (5-CPA) ammonium salt (prepared from enzymatichydrolysis of adiponitrile; filtered product mixture from Example 26),from 0M to 2.5M ammonium hydroxide, and 5 wt. % of Cr-promoted Raneynickel catalyst (Raney 2400) (relative to weight of 5-cyanopentanoicacid) were run in glass shaker tubes at 500 psig hydrogen gas and attemperatures of from 70° C. to 160° C. for 2 h, then analyzed by highpressure liquid chromatography for conversion of 5-CPA and by gaschromatography for the production of caprolactam:

    ______________________________________           5-CPA           ammo-           nium    Temp.  salt      NH.sub.4 OH!                             Time  5-CPA  caprolactam    (° C.)           (M)      (M)      (h)   (% conv.)                                          (% yield)    ______________________________________    70     1.0      0        2     100    0.7    70     1.0      1.0      2     100    0.7    70     1.0      1.5      2     94     0.8    70     1.0      2.0      2     84     0.8    70     1.0      2.5      2     99     0.8    120    1.0      0        2     99     0.9    120    1.0      1.0      2     100    0.9    120    1.0      1.5      2     97     1.0    120    1.0      2.0      2     97     1.1    160    1.0      0        2     97     2.5    160    1.0      1.0      2     84     2.3    160    1.0      1.5      2     97     3.0    160    1.0      2.0      2     95     2.7    ______________________________________

The cyclization of the ammonium salt of 6-aminocaproic acid (6-ACA) inthe aqueous product mixtures prepared by the hydrogenation of 5-CPAammonium salt at 70° C. (as described above) was attempted at a highertemperature by first removing the hydrogenation catalyst by filtration,then heating the ca. 1.0M 6-ACA ammonium salt reaction mixtures at 280°C. for 2 h:

    ______________________________________          6-ACA ammonium salt    Temp. (M) (prepared as                           NH.sub.4 OH!                                   Time caprolactam    (° C.)          described above)                          (M)      (h)  (% yield)    ______________________________________    280   1.0             0        2    17.8    280   1.0             1.0      2    17.2    280   1.0             1.5      2    11.0    280   1.0             2.0      2    9.0    280   1.0             2.5      2    3.1    ______________________________________

EXAMPLE 38 1,5-Dimethyl-2-Piperidone from 4-Cyanopentanoic Acid AmmoniumSalt

Hydrogenations of 5-mL aqueous reaction mixtures containing 1.0M4-cyanopentanoic acid ammonium salt (filtered product mixture fromExample 9, reaction #5), 3.0M methylamine, and 5 wt. % or 10 wt. %(relative to weight of 4-cyanopentanoic acid) of a hydrogenationcatalyst selected from a group consisting of Cr-promoted Raney nickelcatalyst (Raney 2400), 5% palladium on carbon, 5% palladium on alumina,5% ruthenium on alumina, or 4.5% palladium/0.5% platinum on carbon wererun in glass shaker tubes at 500 psig hydrogen gas and at 160° C. for 2h, then analyzed for the production of 1,5-dimethyl-2-piperidone(5-DMPD) and 5-methyl-2-piperidone (5-MPPD):

    ______________________________________                     wt. %       4-CPA    Temp.            cata-  Time (%    5-DMPD 5-MPPD    (° C.)          catalyst   lyst   (h)  conv.)                                       (% yield)                                              (% yield)    ______________________________________    160   Raney 2400 5      2    100   19.2   68.5    160   Raney 2400 10     2    100   21.7   69.1    160   5% Ru/Al2O3                     5      2    100   19.0   63.5    160   5% Pd/C    5      2    99    81.7   7.8    160   5% Pd/Al2O3                     5      2    93    77.2   4.6    160   4.5% Pd/0.5%                     5      2    97    77.8   6.2          Pt/C    ______________________________________

EXAMPLE 39 1,5-Dimethyl-2-Piperidone from 4-Cyanopentanoic Acid AmmoniumSalt

Hydrogenations of 5-mL aqueous reaction mixtures containing 1.0M4-cyanopentanoic acid ammonium salt (filtered product mixture fromExample 9, reaction #5), from 1.0M to 3.0M methylamine, and 5 wt. %(relative to weight of 4-cyanopentanoic acid) of 5% palladium on carbonwere run in glass shaker tubes at 500 psig hydrogen gas and at 140° C.for 2 h, then analyzed for the production of 1,5-dimethyl-2-piperidone(5-DMPD), 5-methyl-2-piperidone (5-MPPD), and 2-methylglutaric acid(2-MGA):

    __________________________________________________________________________    Temp.    time      4-CPA                            5-DMPD                                 5-MPPD                                      2-MGA    (° C.)        catalyst             (h)                 CH3NH.sub.2 !(M)                       (% conv.)                            (% yield)                                 (% yield)                                      (% yield)    __________________________________________________________________________    140 5% Pd/C             2  1.0    100  53.3 0    0.7    140 5% Pd/C             2  1.25   100  65.8 0    0.7    140 5% Pd/C             2  1.5    99   74.5 0    0.8    140 5% Pd/C             2  2.0    98   80.2 0    1.0    140 5% Pd/C             2  3.0    99   83A  0    1.1    __________________________________________________________________________

EXAMPLE 40 5-Dimethyl-2-Piperidone from 4-Cyanopentanoic Acid AmmoniumSalt

Hydrogenations of 5-mL aqueous reaction mixtures containing 1.0M4-cyanopentanoic acid ammonium salt (filtered product mixture fromExample 9, reaction #5), from 1.0M to 4.0M methylamine, and 5 wt. %(relative to weight of 4-cyanopentanoic acid) of 5% palladium on carbonwere run in glass shaker tubes at 500 psig hydrogen gas and at 160° C.for 2 h, then analyzed for the production of 1,5-dimethyl-2-piperidone(5-DMPD), 5-methyl-2-piperidone (5-MPPD), and 2-methylglutaric acid(2-MGA):

    __________________________________________________________________________    Temp.    time                 CH.sub.3 NH.sub.2 !                     4-CPA                          5-DMPD                               5-MPPD                                    2-MGA    (° C.)        catalyst             (h)                (M)  (% conv.)                          (% yield)                               (% yield)                                    (% yield)    __________________________________________________________________________    160 5% Pd/C             2  1.0  100  53.5 0    0.7    160 5% Pd/C             2  1.25 100  68.3 0    0.8    160 5% Pd/C             2  1.5  100  76.4 0    0.9    160 5% Pd/C             2  2.0  100  81.5 0    1.8    160 5% Pd/C             2  3.0  99   81.7 7.8  3.3    160 5% Pd/C             2  4.0  99   88.4 1.9  2.6    __________________________________________________________________________

EXAMPLE 41 1,5-Dimethyl-2-Piperidone from 4-Cyanopentanoic Acid AmmoniumSalt

Hydrogenations of 5-mL aqueous reaction mixtures containing 1.0M4-cyanopentanoic acid ammonium salt (filtered product mixture fromExample 9, reaction #5), from 3.0M to 4.0M methylamine, and 5 wt. %(relative to weight of 4-cyanopentanoic acid) of either 5% palladium oncarbon or 4.5% palladium/0.5% platinum on carbon were run in glassshaker tubes at 500 psig hydrogen gas and at 180° C. for 2 h, thenanalyzed for the production of 1,5-dimethyl-2-piperidone (5-DMPD),5-methyl-2-piperidone (5-MPPD), and 2-methylglutaric acid (2-MGA):

    __________________________________________________________________________    Temp          CH.sub.3 NH.sub.2 !                      4-CPA                           5-DMPD                                5-MPPD                                     2-MGA    (° C.)        catalyst (M)  (% conv.)                           (% yield)                                (% yield)                                     (% yield)    __________________________________________________________________________    180 5% Pd/C  3.0  97   73.0 6.6  --    180 4.5% Pd/0.5% Pt/C                 3.0  96   69.0 1.9  23.9    180 5% Pd/C  4.0  95   66.6 2.6  33.5    __________________________________________________________________________

EXAMPLE 42 1,5-Dimethyl-2-Piperidone from 4-Cyanopentanoic Acid AmmoniumSalt

Into a 100 mL graduated cylinder was placed 54.4 mL of an aqueousreaction mixture containing 1.84M 4-cyanopentanoic acid ammonium salt(0.1 mole 4-cyanopentanoic acid ammonium salt, produced by the enzymatichydrolysis of 2-methylglutaronitrile; Example 9, filtered productmixture from reaction #5), then 25.8 ml of 40 wt. % methylamine (9.31 gmethylamine, 0.3 mole) was added and the final volume adjusted to 100 mLwith distilled water. The final concentrations of 4-cyanopentanoic acidammonium salt and methylamine were 1.0M and 3.0M, respectively. To theresulting solution was added 0.636 g (5 wt. %/wt. of 4-cyanopentanoicacid) of 5% Pd on carbon powder, and the resulting mixture charged to a300-mL 314 SS Autoclave Engineers EZE-Seal stirred autoclave equippedwith a Dispersimax® turbine-type impeller. After flushing the reactorwith nitrogen, the contents of the reactor were stirred at 1000 rpm andheated at 160° C. under 500 psig of hydrogen gas for 4 h. Samples (ca.1.5 mL) were removed via a sampling tube over the course of the reactionfor analysis. After cooling to room temperature, analysis of the finalreaction mixture by gas chromatography indicated a 72.8% yield of1,5-dimethyl-2-piperidone, 3.5% 5-methyl-2-piperidone, 19.9%2-methylglutaric acid, and no 4-cyanopentanoic acid ammonium saltremaining.

The product mixture (61 mL after sampling) was filtered to remove thecatalyst, then adjusted to pH 7.0 with 6N HCl and saturated with sodiumchloride. The resulting solution was extracted four times with 100 mL ofethyl ether. and the combined organic extracts dried over magnesiumsulfate. filtered. and the solvent removed by rotary evaporation underreduced pressure to yield a colorless liquid. This liquid was distilledat 3.5 Torr and the fraction boiling at 70.0-71.5° C. collected to yield4.65 g (60% isolated yield) of 1,5-dimethyl-2-piperidone (5-DMPD).

EXAMPLE 43 4-Ethyl-1-Methylpyrrolidin-2-one from 3-Cyanopentanoic AcidAmmonium Salt

Into a 100 mL graduated cylinder was placed 79.4 mL of an aqueousreaction mixture containing 1.26M 3-cyanopentanoic acid ammonium salt(0.1 mole 3-cyanopentanoic acid ammonium salt, produced by the enzymatichydrolysis of 2-ethylsuccinonitrile; Example 13 filtered productmixture), then 17.2 mL of 40 wt. % methylamine (6.21 g methylamine, 0.2mole) was added and the final volume adjusted to 100 mL with distilledwater. The final concentrations of 3-cyanopentanoic acid ammonium saltand methylamine were 1.0M and 2.0M, respectively. To the resultingsolution was added 0.636 g (5 wt. %/wt. of 3-cyanopentanoic acid) of 5%Pd on carbon powder, and the resulting mixture charged to a 300-mL 314SS Autoclave Engineers EZE-Seal stirred autoclave equipped with aDispersimax® turbine-type impeller. After flushing the reactor withnitrogen, the contents of the reactor were stirred at 1000 rpm andheated at 140° C. under 500 psig of hydrogen gas for 4 h. Samples (ca.1.5 mL) were removed via a sampling tube over the course of the reactionfor analysis. After cooling to room temperature, analysis of the finalreaction mixture by gas chromatography indicated a 69.8% yield of4-ethyl-1-methylpyrrolidin-2-one and a 20.4% yield of4-ethylpyrrolidin-2-one, with no 3-cyanopentanoic acid ammonium saltremaining.

The product mixture (80 mL after sampling) was filtered to remove thecatalyst, then adjusted to pH 7.0 with 6N HCl and saturated with sodiumchloride. The resulting solution was extracted four times with 100 mL ofdichloro-methane, and the combined organic extracts dried over magnesiumsulfate, filtered, and the solvent removed by rotary evaporation underreduced pressure to yield a colorless liquid. This liquid wasfractionally-distilled at 16 Torr, and the fraction boiling at 100° C.was collected (5.15 g, 51% yield). The resulting4-ethyl-1-methylpyrrolidin-2-one pyrrolidin-2-one contained <5%4-ethylpyrrolidin-2-one as impurity, so the liquid was redistilled at 40Torr and the fraction boiling at 128° C. collected to yield 3.71 g (37%isolated yield) of 4-ethyl-1-methylpyrrolidin-2-one (4-EMPRD).

EXAMPLE 44 5-Methyl-2-Piperidone from 4-Cyano-4-Pentenoic Acid AmmoniumSalt

Into a 100 mL graduated cylinder was placed 77.0 mL of an aqueousreaction mixture containing 1.30M 4-cyano-4-pentenoic acid ammonium salt(0.1 mole 4-cyanopentanoic acid ammonium salt, produced by the enzymatichydrolysis of 2-methyleneglutaronitrile; Example 17), then 12.9 mL ofconcentrated ammonium hydroxide (29.3% NH₃, 0.2 mole NH₃) was added andthe final volume adjusted to 100 mL with distilled water. The finalconcentrations of 4-cyano-4-pentenoic acid ammonium salt and addedammonium hydroxide were 1.0M and 2.0M, respectively. To the resultingsolution was added 0.626 g (5 wt. %/wt. of 4-cyano-4-pentenoic acid) ofchromium-promoted Raney Nickel (Grace Davison Raney® 2400 Active MetalCatalyst), and the resulting mixture charged to a 300-mL 314 SSAutoclave Engineers EZE-Seal stirred autoclave equipped with aDispersimax® turbine-type impeller. After flushing the reactor withnitrogen, the contents of the reactor were stirred at 1000 rpm andheated under 500 psig of hydrogen gas at 50° C. for 5 h, then for anadditional 3 h at 160° C. After cooling to room temperature, analysis ofthe final reaction mixture by gas chromatography indicated a 85.0% yieldof 5-methyl-2-piperidone, with no 4-cyano-4-pentenoic acid ammonium saltremaining.

EXAMPLE 45 2-Pyrrolidinone from 3-Cyanopropionic Acid Ammonium Salt

Into a 100 mL graduated cylinder was placed 75.8 mL of an aqueousreaction mixture containing 1.31M 3-cyanopropionic acid ammonium salt(0.1 mole 3-cyanopropionic acid ammonium salt, produced by the enzymatichydrolysis of succinonitrile; Example 20), then 19.4 mL of concentratedammonium hydroxide (29.3% NH₃, 0.3 mole NH₃) was added and the finalvolume adjusted to 100 mL with distilled water. The final concentrationsof 3-cyanopropionic acid ammonium salt and added ammonium hydroxide were1.0M and 3.0M, respectively. To the resulting solution was added 0.99 g(10 wt. %/wt. of 3-cyanopropionic acid) of chromium-promoted RaneyNickel (Grace Davison Raney® 2400 Active Metal Catalyst), and theresulting mixture charged to a 300-mL 314 SS Autoclave EngineersEZE-Seal stirred autoclave equipped with a Dispersimax® turbine-typeimpeller. After flushing the reactor with nitrogen, the contents of thereactor were stirred at 1000 rpm and heated under 500 psig of hydrogengas at 70° C. for 4.5 h, then for an additional 5 h at 180° C. Analysisby gas chromatography indicated a 91.0% yield of 2-pyrrolidinone, withno 3-cyanopropionic acid ammonium salt remaining.

EXAMPLE 46 2-Piperidone from 4-Cyanobutyric Acid Ammonium Salt

The procedure described in Example 22 was repeated. After 4.0 h, theHPLC yield of 4-cyanobutyric acid ammonium salt and glutaric aciddiammonium salt was 91.7% and 7.5%, respectively, with no glutaronitrileremaining. The final concentration of 4-cyanobutyric acid ammonium saltin the centrifuged and filtered reaction mixture was 1.42M. Into a 100mL graduated cylinder was placed 70.6 mL (0.100 mole of 4-cyanobutyricacid ammonium salt) of the filtered aqueous reaction mixture, then 19.4mL of concentrated ammonium hydroxide (29.3% NH₃, 0.3 mole NH₃) wasadded and the final volume adjusted to 100 mL with distilled water. Thefinal concentrations of 4-cyanopropionic acid ammonium salt and addedammonium hydroxide were 1.0M and 3.0M, respectively. To the resultingsolution was added 1.13 g (10 wt. %/wt. of 4-cyanobutyric acid) ofchromium-promoted Raney Nickel (Grace Davison Raney® 2400 Active MetalCatalyst), and the resulting mixture charged to a 300-mL 314 SSAutoclave Engineers EZE-Seal stirred autoclave equipped with aDispersimax® turbine-type impeller. After flushing the reactor withnitrogen, the contents of the reactor were stirred at 1000 rpm andheated under 500 psig of hydrogen gas at 70° C. for 3.5 h. Analysis bygas chromatography indicated a 29.7% yield of 2-piperidone, with no4-cyanobutyric acid ammonium salt remaining. The temperature wasincreased to 180° C. for an additional 2 h, and subsequent analysis of asample by gas chromatography indicated a 93.5% yield of 2-pyrrolidinone.

EXAMPLE 47 5-Methyl-2-Piperidone from 4-Cyanopentanoic Acid AmmoniumSalt

Fifty milliliters of an aqueous mixture containing 1.85M4-cyanopentanoic acid ammonium salt (0.1 mole 4-cyanopentanoic acidammonium salt, produced by the enzymatic hydrolysis of2-methylglutaronitrile; Example 9, filtered product mixture fromreaction #5), 5.6 g of 29% aqueous ammonium hydroxide and 44 mL D.I.water was charged to a 300 mL autoclave. To this solution was added 0.73g (3 wt% based on 4-cyanopentanoic acid) of 4.5% Pd/0.5% Pt on carboncatalyst. The autoclave was sealed and purged 3 times with hydrogenfollowed by heating to 160° C. under 100 psig hydrogen and slowstirring. At 160° C., the pressure was raised to 800 psig and maximumstirring commenced. After 3 hours, the reactor was cooled, vented andpurged with nitrogen. Gas chromatographic analysis of the productmixture indicated a 96% conversion of 4-cyanopentanoic acid and a 95.5%yield (99.5% selectivity) to 5-methyl-2-piperidone.

EXAMPLE 48 1,5-Dimethyl-2-Piperidone from 4-Cyanopentanoic Acid AmmoniumSalt

One hundred milliliters of an aqueous mixture containing 1.85M4-cyanopentanoic acid ammonium salt (0.2 mole 4-cyanopentanoic acidammonium salt, produced by the enzymatic hydrolysis of2-methylglutaronitrile; Example 9, filtered product mixture fromreaction #5), and 14 g (0.2 mole) 40% aqueous methylamine was charged toa 300 mL autoclave. To this solution was added 4.5% Pd/0.5% Pt on carboncatalyst. The autoclave was sealed and purged 3 times with hydrogenfollowed by heating to reaction under 100 psig hydrogen and slowstirring. At reaction temperature, the pressure was raised and maximumstirring commenced. After a given reaction time, the reactor was cooled,vented and purged with nitrogen. Gas chromatographic analysis of theproduct mixture for several runs at different reaction conditions aresummarized below:

    ______________________________________                 catalyst    Temp. H.sub.2                 loading   time 4-CPA  5-DMPD 5-MPPD    (° C.)          (psig) (wt %)    (h)  (% conv.)                                       (% yield)                                              (% yield)    ______________________________________    175   300    7.4       3    99     67.8   28.2    160   500    10        2    99     68.6   28.1    145   500    10        2    93     65.2   26.4    ______________________________________

EXAMPLE 49 1,5-Dimethyl-2-Piperidone from 4-Cyanopentanoic Acid AmmoniumSalt

Hydrogenations of 4-cyanopentanoic acid ammonium salt were performed in5 mL glass shaker tubes at 800 psig using different Pd on carboncatalysts. Four milliliters of 1.85M (7 mmoles) aqueous 5-cyanopentanoicacid (filtered product mixture from Example 9, reaction #5), 0.88 g(11.4 mmoles) 40% methylamine and catalyst from a group consisting of 5%Pd/C and 4.5% Pd/0.5% Pt/C were charged to the tube and run for 3 hours.Gas chromatographic analysis of the product mixtures, after cooling to25°, at different reaction conditions are summarized below:

    ______________________________________                   catalyst    Temp.          loading time 4-CPA  5-DMPD S-MPPD    (° C.)          catalyst (wt %)  (h)  (% conv.)                                       (% yield)                                              (% yield)    ______________________________________    160   5% Pd/C  1.2     2    99     91.8   5.0    160   4.5% Pd/ 0.7     2    99     94.0   3.1          0.5% Pt/C    150   5% Pd/C  0.7     2    99     92.5   2.2    ______________________________________

We claim:
 1. A compound of Formula III, ##STR6## where M⁺ is either H⁺or NH₄ ⁺.